U.S. patent number 7,270,730 [Application Number 10/236,684] was granted by the patent office on 2007-09-18 for high-throughput electrophysiological measurement system.
This patent grant is currently assigned to Essen Instruments, Inc.. Invention is credited to Bradley D. Neagle, Kirk S. Schroeder.
United States Patent |
7,270,730 |
Schroeder , et al. |
September 18, 2007 |
High-throughput electrophysiological measurement system
Abstract
Systems, including apparatus and methods, for performing
electrophysiological measurements on membranous samples, including
living cells, isolated cell fragments (such as organelles), and/or
artificial membranes (such as vesicles). The apparatus may include
a high-throughput electrophysiological measurement system, and
components thereof. This measurement system may include, among
others, (1) a fluidics head for transferring samples and/or other
compounds to a perforated measurement substrate, (2) a
pressure-regulated plenum system for positioning samples on the
substrate and subsequently forming a high-resistance electrical
seal, (3) an activation system (such as a computer-controlled
pulsed UV illumination module) for activating caged compounds, (4)
an electronics head for applying and/or measuring voltage and/or
current, and/or (5) a computer-controlled analysis system for
collecting and/or analyzing data. The methods may include methods
for performing high-throughput electrophysiological measurements on
transporters and/or voltage or ligand-gated ion channels,
sequentially and/or simultaneously.
Inventors: |
Schroeder; Kirk S. (Ann Arbor,
MI), Neagle; Bradley D. (Ann Arbor, MI) |
Assignee: |
Essen Instruments, Inc. (Ann
Arbor, MI)
|
Family
ID: |
27502132 |
Appl.
No.: |
10/236,684 |
Filed: |
September 5, 2002 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20030070923 A1 |
Apr 17, 2003 |
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Related U.S. Patent Documents
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Application
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Filing Date |
Patent Number |
Issue Date |
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PCT/US02/16122 |
May 21, 2002 |
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09862056 |
May 21, 2001 |
7067046 |
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09631909 |
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6488829 |
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60383196 |
May 22, 2002 |
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60317112 |
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Current U.S.
Class: |
204/403.01;
435/287.1 |
Current CPC
Class: |
G01N
33/48728 (20130101); G01N 35/10 (20130101); G01N
2035/00237 (20130101); G01N 2035/1034 (20130101) |
Current International
Class: |
G01N
33/487 (20060101) |
Field of
Search: |
;204/403.01
;435/287.1,287.5,817 ;422/63,65 |
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|
Primary Examiner: Olsen; Kaj K.
Attorney, Agent or Firm: Morgan, Lewis & Bockius LLP
Brezner; David J. Johnson; Victor E.
Parent Case Text
CROSS-REFERENCES TO RELATED APPLICATIONS
This application is a continuation-in-part of the following patent
applications: U.S. patent application Ser. No. 09/631,909, filed
Aug. 4, 2000, now U.S. Pat. No. 6,488,829; U.S. patent application
Ser. No. 09/862,056, filed May 21, 2001, now U.S. Pat. No.
7,067,046; and PCT Patent Application Serial No. PCT/US02/16122,
filed May 21, 2002. This application also claims the benefit under
35 U.S.C. .sctn. 119(e) of the following U.S. provisional patent
applications: Ser. No. 60/317,112, filed Sep. 6, 2001; and Ser. No.
60/383,196, filed May 22, 2002.
U.S. patent application Ser. No. 09/631,909, in turn, claims the
benefit under 35 U.S.C. .sctn. 119(e) of the following U.S.
provisional patent applications: Ser. No. 60/147,253, filed Aug. 5,
1999; and Ser. No. 60/176,698, filed Jan. 18, 2000.
U.S. patent application Ser. No. 09/862,056, in turn, is a
continuation-in-part of U.S. patent application Ser. No.
09/631,909, which, in turn, claims priority directly from two U.S.
provisional patent applications, as indicated above.
PCT Patent Application Serial No. PCT/US02/16122, in turn, is a
continuation-in-part of U.S. patent application Ser. No.
09/862,056, filed May 21, 2001, which, in turn, claims priority
directly and indirectly from several U.S. and provisional patent
applications, as indicated above.
The above-identified U.S., PCT, and provisional priority patent
applications are all incorporated herein by reference in their
entirety for all purposes.
Claims
We claim:
1. Electrophysiological measurement apparatus, comprising: a
multi-well plate having a plurality of fluid chambers, each
configured to support a biological material to be measured; a thin
substrate having an array of apertures in alignment with the
chambers of the multi-well plate, wherein the substrate is joined
to the multi-well plate such that the chambers are open at the top
and sealed at the bottom, except for the apertures, and wherein the
diameter of the apertures is less than the diameter of the
biological material, thereby enabling a high-resistance seal to be
formed between the biological material in each chamber and a
corresponding aperture; a fluid plenum to receive the multi-well
plate such that one side of the substrate is immersed; a transfer
device configured to move material into the fluid chambers of the
multi-well plate while it is in contact with the fluid plenum: a
first electrode disposed in the fluid plenum; at least one second
electrode that can be selectively connected with and disconnected
from the fluid chambers of the multi-well plate, wherein the second
electrode disconnects from the fluid chambers when the transfer
device moves material into the fluid chambers; and
electrophysiological measurement circuitry in electrical
communication with the electrodes; wherein the substrate is
selected from the group consisting of polyethylene terephthalate
(PET) and polyimide and further wherein the substrate has a glass
coating at least in the region where the high-resistance seal is
formed between the material and the substrate.
2. The apparatus of claim 1, wherein there is a single aperture
associated with each chamber of the multi-well plate.
3. The apparatus of claim 1, wherein each fluid chamber contains a
biological material to be measured.
4. The apparatus of claim 1, wherein the diameter of the apertures
is in the range of about 1 to 10 micrometers.
5. The apparatus of claim 1, wherein the apertures are tapered.
6. The apparatus of claim 1, wherein the multi-well plate is sealed
to the fluid plenum, enabling a differential pressure to be applied
relative to the fluid in each chamber, thereby causing the material
in each chamber to migrate to a respective aperture.
7. The apparatus of claim 1, wherein the multi-well plate is sealed
to the fluid plenum, enabling a differential pressure to be
maintained relative to the fluid in each chamber until between the
material in each chamber forms the high-resistance seal to the
corresponding aperture.
8. The apparatus of claim 1, wherein the fluid plenum includes a
chemical reagent causing the material in each chamber to
electrically permeabilize in the vicinity of the aperture.
9. The apparatus of claim 1, wherein a high voltage is temporarily
applied across the electrodes to permeabilize the material in each
chamber, at least in the vicinity of the apertures.
10. The apparatus of claim 1, further comprising a mechanism for
moving the electrode into the chambers of the multi-well plate so
as to automate the measurement of the material contained
therein.
11. The apparatus of claim 1, wherein the at least one second
electrode is moveable into and out of the top openings of the fluid
chambers of the multi-well plate.
12. The apparatus of claim 1, further comprising a plurality of
electrodes in alignment with a plurality of the chambers of the
multi-well plate.
13. The apparatus of claim 12, further comprising a mechanism for
moving the electrodes into the chambers of the multi-well plate to
perform simultaneous measurements on the material contained
therein.
14. The apparatus of claim 1, further comprising a system for
transferring fluids from one or more sources to the chambers of the
multi-well plate.
15. The apparatus of claim 1, wherein the second electrode is
configured to move out from the fluid chambers to disconnect from
the fluid chambers.
16. Electrophysiological measurement apparatus, comprising: a
multi-well plate having a plurality of fluid chambers, each
configured to support a biological material to be measured; a thin
substrate having an array of apertures in alignment with the
chambers of the multi-well plate, wherein the substrate is joined
to the multi-well plate such that the chambers are open at the top
and sealed at the bottom, except for the apertures, and wherein the
diameter of the apertures is less than the diameter of the
biological material, thereby enabling a high-resistance seal to be
formed between the biological material in each chamber and a
corresponding aperture; a fluid plenum to receive the multi-well
plate such that one side of the substrate is immersed; a first
electrode disposed in the fluid plenum; at least one second
electrode that can be connected with and disconnected from the
fluid chambers of the multi-well plate; and electrophysiological
measurement circuitry in electrical communication with the
electrodes, wherein the substrate is a plastic substrate having a
glass coating at least in the region where the high-resistance seal
is formed between the material and the substrate, wherein the
substrate is selected from the group consisting of polyethylene
terephthalate (PET) and polyimide.
17. The apparatus of claim 16, further comprising a fluidics system
operative to control and regulate the differential pressure across
the substrate to achieve a high-resistance electrical seal between
the substrate and the biological material.
18. The apparatus of claim 17, wherein the fluidics system is
further operative to remove trapped gas from both sides of the
substrate to form a continuous fluid pathway for conducting
electrical current.
19. The apparatus of claim 17, wherein the fluidics system is
further operative to apply a vacuum so as to pneumatically isolate
a region on one side of the substrate.
20. The apparatus of claim 16, further comprising reagent inputs
for at least one of an extracellular saline solution, an
intracellular saline solution, a wash solution, and a chemically
altered intracellular saline solution used to achieve low
resistance electrical access to the inside of a cell.
21. The apparatus of claim 20, wherein intracellular solutions are
exchanged without introducing pressure changes that would disrupt
the high-resistance electrical seal.
22. The apparatus of claim 16, further comprising a signal
processor in communication with the electrodes, wherein the signal
processor includes a multiplexer operative to route electrical
signals derived from one or more of the electrodes on a selective
basis.
23. The apparatus of claim 22, wherein the signal processor
includes a low-noise, high-gain trans-impedance operational
amplifier circuit and one or more isolated recording amplifier
circuits.
24. The apparatus of claim 16, further comprising a moveable
electronics head equipped with one or more sensing electrodes in
communication with a signal processor, a moveable fluidics head
equipped with one or more fluid-dispensing needles, the apparatus
further including one or more positions for washing for the
electronics head and/or the fluidics head.
25. The apparatus of claim 24, wherein each position for washing is
automatically filled and drained via a peristaltic pump and
vacuum-assisted waste line.
26. The apparatus of claim 24, wherein each position for washing is
capable of washing an entire head or portions thereof to conserve
conserving wash solution.
27. The apparatus of claim 24, further comprising one or more
compound microplates, each having a standard well format accessible
by the fluidics head.
28. The apparatus of claim 27, further comprising one or more
saline solution reservoirs accessible by the fluidics head.
29. The apparatus of claim 24, further comprising a removable boat
station for cell slurry addition accessible by the fluidics
head.
30. The apparatus of claim 16, further comprising an electronics
head using silver sensing electrodes, wherein the apparatus
includes a station accessible by the electronics head used for
depositing chloride on the electrodes.
31. The apparatus of claim 16, wherein there is a single aperture
associated with each chamber of the multi-well plate.
32. The apparatus of claim 16, wherein each fluid chamber contains
a biological material to be measured.
33. The apparatus of claim 16, wherein the substrate has a glass
coating at least in the region where the high-resistance seal is
formed between the material and the substrate.
34. The apparatus of claim 16, wherein the diameter of the
apertures is in the range of about 1 to 10 micrometers.
35. The apparatus of claim 16, wherein the apertures are
tapered.
36. The apparatus of claim 16, wherein the multi-well plate is
sealed to the fluid plenum, enabling a differential pressure to be
applied relative to the fluid in each chamber, thereby causing the
material in each chamber to migrate to a respective aperture.
37. The apparatus of claim 16, wherein the multi-well plate is
sealed to the fluid plenum, enabling a differential pressure to be
maintained relative to the fluid in each chamber until between the
material in each chamber forms the high-resistance seal to the
corresponding aperture.
38. The apparatus of claim 16, wherein the fluid plenum includes a
chemical reagent causing the material in each chamber to
electrically permeabilize in the vicinity of the aperture.
39. The apparatus of claim 16, wherein a high voltage is
temporarily applied across the electrodes to permeabilize the
material in each chamber, at least in the vicinity of the
apertures.
40. The apparatus of claim 16, further comprising a mechanism for
moving the electrode into the chambers of the multi-well plate so
as to automate the measurement of the material contained
therein.
41. The apparatus of claim 16, wherein the at least one second
electrode is moveable into and out of the top openings of the fluid
chambers of the multi-well plate.
42. The apparatus of claim 16, further comprising a plurality of
electrodes in alignment with a plurality of the chambers of the
multi-well plate.
43. The apparatus of claim 42, further comprising a mechanism for
moving the electrodes into the chambers of the multi-well plate to
perform simultaneous measurements on the material contained
therein.
44. The apparatus of claim 16, further comprising a system for
transferring fluids from one or more sources to the chambers of the
multi-well plate.
Description
CROSS-REFERENCES TO ADDITIONAL MATERIALS
This application incorporates by reference in their entirety for
all purposes the following publications: Richard P. Haugland,
HANDBOOK OF FLUORESCENT PROBES AND RESEARCH CHEMICALS (6.sup.th ed.
1996); and Joseph R. Lakowicz, PRINCIPLES OF FLUORESCENCE
SPECTROSCOPY (2.sup.nd ed. 1999).
FIELD OF THE INVENTION
The invention relates to electrophysiology. More particularly, the
invention relates to systems for performing electrophysiological
measurements, typically in parallel, and typically without direct
human intervention, especially to understand the properties and/or
interactions of specific membrane components, such as ligand-gated
ion channels and/or transporters.
BACKGROUND OF THE INVENTION
The electrical behavior of cells and cell membranes is of profound
importance in basic research as well as in modern drug development.
A specific area of interest in this field is in the study of ion
channels and transporters [1]. Ion channels are protein-based pores
found in the cell membrane that are responsible for maintaining the
electrochemical gradients between the extracellular environment and
the cell cytoplasm. These channels quite often are selectively
permeable to a particular type of ion, e.g., calcium, chloride,
potassium, or sodium. The channels generally comprise two parts:
(1) the pore itself, and (2) a switch mechanism that regulates the
conductance of the pore. The switch mechanism may be controlled by
transmembrane voltage changes, covalent modification, mechanical
stimulation, and/or chemical ligands (e.g., through the activation
or deactivation of an associated membrane receptor), among others.
Ion channels are passive elements in that, once opened, ions flow
in the direction of existing electrochemical gradients. Ion
transporters are similar to ion channels in that they are involved
in the transport of ions across cell membranes; however, they
differ from ion channels in that they require energy for their
function and in that they tend to pump actively against established
electrochemical gradients.
Ion channels are prevalent in the body and are necessary for many
physiological functions, including the beating of the heart, the
contraction of voluntary muscles, and the signaling of neurons.
They also are found in the linings of blood vessels, allowing for
physiological regulation of blood pressure, and in the pancreas,
allowing for the control of insulin release. As such, the study of
ion channels is a very diverse and prolific area encompassing basic
academic research as well as biotechnical and pharmaceutical
research. Experiments on ion channels may be performed on cell
lines that endogenously express the ion channel of interest
("native channels") as well as on recombinant expression systems
such as the Xenopus oocyte or mammalian cell lines (e.g., CHO, HEK,
etc.) that have been transiently or stably transfected to express
the ion channel by well-known techniques [2, 3]. Electrophysiology
also is performed on isolated cell membranes or vesicles as well as
on synthetic membranes where solubilized channels are reconstituted
into a manufactured membrane [4].
I. Instrumentation
To date, the most useful and widely utilized tool for the study of
ion channels and transporters is a technique called "patch
clamping." This technique was first introduced almost 25 years ago
[5-7], and consists of using a small glass capillary to function as
an electrode in measuring currents and voltages from individual
cells. FIG. 1 shows a typical patch clamp measurement geometry. A
glass capillary 2 is first heated and pulled to a fine tip. The
capillary is then filled with a saline buffer solution 4 and fitted
with a Ag/AgCl electrode 6. The function of the Ag/AgCl electrode
is to provide an electrical connection to a wire via the reversible
exchange of chloride ions in the pipette solution.
Through the use of a microscope and micromanipulating arm (not
shown), the user finds a biological cell or cell membrane 8
containing ion channels 10 of interest and gently touches the cell
membrane with the pipette. The measurement circuit is completed via
the external ionic solution 12 and a second Ag/AgCl bath electrode
14. A high-impedance operational amplifier 16 senses the current
flowing in the circuit, which is subsequently recorded and analyzed
with a data recording system 18. A key to the successful function
of the technique is the ability to form a high electrical
resistance (.about.1 G.OMEGA.) seal between the glass pipette and
the cell membrane 20, so that the current recorded by the amplifier
is dominated by ions 22 flowing through the cell membrane and not
by ions flowing around the glass pipette directly into the bath
solution.
Once a high-resistance seal is achieved between the pipette and the
cell membrane, there are many measurement configurations that the
system can take, including the "whole-cell," "perforated-patch,"
and "inside-out" patch clamp configurations. The whole-cell voltage
clamp is one of the more common configurations. In the whole-cell
voltage clamp, the portion of membrane at the end of the pipette 24
is permeabilized so as effectively to place the pipette electrode
inside the cell. This, in turn, allows for an external voltage
command 26 to be placed between the intracellular pipette electrode
and the extracellular bath electrode, thereby providing control of
the cell's transmembrane voltage potential. The term "whole cell"
is derived from the fact that, with this configuration, the
instrument measures the majority of the currents in the entire cell
membrane.
The electrical permeabilization of the membrane at the end of the
pipette can be induced in many ways. Permeabilization often is
achieved by using voltage pulses of sufficient strength and
duration that the membrane inside the pipette physically breaks
down. This approach is well known in the field and is commonly
referred to as "zapping" [8]. Permeabilization also may be achieved
by using certain antibiotics, such as Nystatin and Amphotericin B
[9]. These antibiotics work by forming chemical pores in the cell
membrane that are permeable to monovalent ions, such as chloride.
Since chloride is the current-carrying ion for the commonly used
Ag/AgCl electrode, these antibiotics can produce a low resistance
electrical access to the interior of the cell. The advantage of the
chemical technique is that the membrane patch remains intact so
that larger intracellular molecules remain inside the cell, rather
than being flushed out by the pipette solution as with the zapping
technique. This approach also is well known in the field and is
commonly referred to as a "perforated patch" [8-10].
The formation of high-resistance electrical seals enables the
measurement system to detect very small physiological membrane
currents (e.g., .about.10.sup.-12 A). In addition, by perforating a
portion of the cell membrane either electrically or chemically, it
is possible to control the voltage (voltage clamp) or current
(current clamp) across the remaining intact portion of the cell
membrane. This greatly enhances the utility of the technique for
making physiological measurements of ion channel/transporter
activity, since quite often this activity is dependent on
transmembrane voltage. By being able to control the trans-membrane
voltage (or current), it is possible to stimulate or deactivate ion
channels or transporters with great precision and as such greatly
enhance the ability to study complex drug interactions.
The development of the patch clamp technique revolutionized the
field of electrophysiology, allowing for the direct electrical
measurement of ion channel/transporter events in living cells, cell
membranes, and artificial membranes. However, existing patch clamp
techniques require operators with high levels of manual dexterity
who must learn to record data from single cell or membrane
preparations using a small glass capillary positioned under a
microscope by a micromanipulating arm. Moreover, even skilled
operators typically require tens of minutes to complete a single
recording session, while, in the case of drug screening, it
generally is preferable to obtain a new cell sample for each
different chemical entity to be tested. Thus, existing techniques
are not capable of looking at thousands of different conditions
(e.g., chemical stimuli) per day, a common need in the biotechnical
or pharmaceutical industry.
U.S. Pat. No. 6,063,260 to Olesen describes a system intended to
improve the throughput and decrease the fluid volume required of
standard patch clamp technology. The improvement relies on using a
standard HPLC autosampler apparatus integrated into a standard
patch clamp arrangement to more easily inject multiple fluids
samples into the measurement system. The invention claims to
increase throughput by making multiple sequential fluid additions
to the same biological membrane faster and easier. However, the
Olesen invention is deficient in several respects. First, it does
not allow for a plurality of different biological samples to be
measured simultaneously. Second, it does not eliminate the
labor-intensive aspects of micromanipulation involved in standard
patch clamp electrophysiology. Third, it does not address cases in
biological drug screening where multiple chemical reagent additions
to the same biological sample are to be avoided (as in the case of
high-throughput drug screening).
Published PCT Application No. WO 99/66329 discusses the use of a
perforated screen to conduct tests on biological materials, but the
proposed system has significant, severe limitations in terms of
practical implementation. For example, all embodiments discussed in
the WO 99/66329 application utilize multiple apertures per fluid
well, placing reliance on the growth of confluent cell matrices to
effectuate sealing of the multiple perforations formed in
relatively thick material. In addition, although the published
application makes reference to automation, no workable, fully
integrated systems are disclosed that are capable of high
throughput and reliability.
The invention may address these and/or other shortcomings by
providing instrumentation for automated, high-throughput studies of
ion channels.
II. Ion Channel Assays
The rapid and diverse signaling kinetics of ion channels makes
their study both interesting and technically challenging. Many ion
channels can be activated and then deactivated in a few
milliseconds. This rapid time scale implies that the
instrumentation used to study channel kinetics should have a fairly
high frequency bandwidth, for example, on the order of 10 kHz.
Fortunately, such bandwidths are attainable, since high-bandwidth
operational amplifiers are readily available. Unfortunately, this
rapid time scale further implies that the method of stimulating ion
channel events also should be fast.
The needed time scale of the stimulus depends in part on whether
the channels are voltage gated or ligand gated. Voltage gated
channels are activated or deactivated by changes in transmembrane
voltage, as mentioned previously. For these channels, the same
electronics used to record ion channel currents also can be used to
control the voltage stimulus, since the time bandwidth of the
stimulus, an electrical signal, is inherently fast enough to avoid
degrading the kinetics of the voltage-gated ion channel signals. In
contrast, ligand-gated channels are activated or deactivated by
chemical or ligand binding. These channels may be gated by specific
chemical messengers, such as the release of intracellular calcium,
adenosine 3',5'-monophosphate (cyclic AMP or cAMP), or
acetylcholine (ACh), among others. In some cases, the chemical
activation of an ion channel is extracellular in its initiation,
and, in other cases, the chemical activation is intracellular. This
implies that it is important that the compound not only can be
released on the time scale of tens of milliseconds, but in some
cases that the compound can be introduced within the membrane of a
living cell.
The invention may address these and/or other shortcomings by
providing channel assays for automated, high-throughput studies of
voltage and/or ligand-gated ion channels.
SUMMARY OF THE INVENTION
The invention provides systems, including apparatus and methods,
for performing electrophysiological measurements on membranous
samples, including living cells, isolated cell fragments (such as
organelles), and/or artificial membranes (such as vesicles). The
apparatus may include a high-throughput electrophysiological
measurement system, and components thereof. This measurement system
may include, among others, (1) a fluidics head for transferring
samples and/or other compounds to a perforated measurement
substrate, (2) a pressure-regulated plenum system for positioning
samples on the substrate and subsequently forming a high-resistance
electrical seal, (3) an activation system (such as a
computer-controlled pulsed UV illumination module) for activating
caged compounds, (4) an electronics head for applying and/or
measuring voltage and/or current, and/or (5) a computer-controlled
analysis system for collecting and/or analyzing data. The methods
may include methods for performing high-throughput
electrophysiological measurements on transporters and voltage or
ligand-gated ion channels, sequentially and/or simultaneously.
BRIEF DESCRIPTION OF DRAWINGS
FIG. 1 is a schematic view of a prior-art patch clamp
electrophysiology configuration, showing measurement geometry.
FIG. 2 is a schematic view of the formation of an electrical seal
between a single cell and a single hole in a substrate, in
accordance with aspects of the invention.
FIG. 3 is a schematic view of aperture geometry, showing two
configurations for seal formation, in accordance with aspects of
the invention.
FIG. 4 is a graph showing command voltage protocol and measured
electrical leak resistance between a transfected CHO cell and a
SiO.sub.2-coated Kapton-film aperture.
FIG. 5 is a graph showing whole cell physiological currents
measured on CHO cells transfected with the voltage-gated potassium
channel Kv3.2, for a voltage sweep from -100 mV to +60 mV, and for
a voltage step protocol from -70 mV to various step voltages.
FIG. 6 is a partially schematic view of an exemplary system
platform layout for conducting high-throughput electrophysiological
measurements, in accordance with aspects of the invention.
FIG. 7 is a picture of the system platform layout of FIG. 6,
showing additional features of the layout.
FIG. 8 is an alternative schematic view of the system platform
layout of FIG. 6, showing additional features of the layout.
FIG. 9 is a picture of a system cabinet and bottle layout for
conducting high-throughput electrophysiological measurements, in
accordance with aspects of the invention.
FIG. 10 is an exploded perspective view of an exemplary membrane
carrier, membrane substrate, and plenum, in accordance with aspects
of the invention.
FIG. 11 is a schematic view of an exemplary plenum fluidics system,
in accordance with aspects of the invention.
FIG. 12 is a schematic view of an exemplary plenum vacuum
regulation system, in accordance with aspects of the invention.
FIG. 13 is a set of views of an exemplary sample-handling fluidics
head, in accordance with aspects of the invention.
FIG. 14 is an exploded partially perspective, partially schematic
view of an exemplary electronics head, in accordance with aspects
of the invention.
FIG. 15 is a schematic view of portions of the system of FIG. 14,
showing the use of two separate banks of electrode pins.
FIG. 16 is a schematic view of an exemplary activation system,
showing how light energy may be directed via optical fibers to a
plurality of biological samples, in accordance with aspects of the
invention.
FIG. 17 is a screen shot from an exemplary graphical user interface
showing 48 time traces from a "hole test" electrical measurement,
in accordance with aspects of the invention
FIG. 18 is a screen shot from an exemplary graphical user interface
showing 48 time traces from a "seal test" electrical measurement,
in accordance with aspects of the invention.
FIG. 19 is a screen shot from an exemplary graphical user interface
showing 48 time traces from a electrophysiological recording of
sodium channels in CHO cells, in accordance with aspects of the
invention.
FIG. 20 is a screen shot from an exemplary graphical user interface
regarding data file naming conventions, directory handling, and
display of experiment summaries, in accordance with aspects of the
invention.
FIG. 21 is a screen shot from an exemplary graphical user interface
regarding the setup and timing of a resistance "seal test," along
with plate usage definitions, in accordance with aspects of the
invention.
FIG. 22 is a screen shot from an exemplary graphical user interface
regarding the setup and timing of the addition of a perforation
agent to the experimental protocol, along with experiment "type"
definitions, in accordance with aspects of the invention.
FIG. 23 is a screen shot from an exemplary graphical user interface
regarding the setup and timing of the command voltage waveform
protocols used in high-throughput electrophysiological recordings,
in accordance with aspects of the invention.
FIG. 24 is a screen shot from an exemplary graphical user interface
regarding the setup and timing of the fluidics head compound
additions and manual voltage offset corrections used in
high-throughput electrophysiological recordings, in accordance with
aspects of the invention.
FIG. 25 is a screen shot from an exemplary graphical user interface
regarding the display of compiled success rates and plate "hits"
for a high-throughput electrophysiological data set, in accordance
with aspects of the invention.
FIG. 26 is a screen shot from an exemplary graphical user interface
showing four electrophysiological traces of sodium currents
corresponding to one non-active compound from a high-throughput
electrophysiological data set, in accordance with aspects of the
invention.
FIG. 27 is a screen shot from an exemplary graphical user interface
regarding the setup and definition processing "metrics" used in
data reduction analysis of high-throughput electrophysiological
recordings, in accordance with aspects of the invention.
FIG. 28 are time traces of electrophysiological data showing the
effects of resistance leak correction on a high-throughput
electrophysiological data trace, in accordance with aspects of the
invention.
DETAILED DESCRIPTION
The invention provides systems, including apparatus and methods,
for performing electrophysiological measurements on membranous
samples, including living cells, isolated cell fragments (such as
organelles), and/or artificial membranes (such as vesicles). The
apparatus may include a high-throughput electrophysiological
measurement system, and components thereof. This measurement system
may include, among others, (1) a fluidics head for transferring
samples and/or other compounds to a perforated measurement
substrate, (2) a pressure-regulated fluidics system for positioning
samples on the substrate and subsequently forming a high-resistance
electrical seal, (3) an activation system (such as a
computer-controlled pulsed UV illumination module) for activating
caged compounds, (4) an electronics head for applying and/or
measuring voltage and/or current, and/or (5) a computer-controlled
analysis system for collecting and/or analyzing data. The methods
may include methods for performing high-throughput
electrophysiological measurements, for example, using activatable
or caged compounds to study ligand-gated ion channels and
transporters, sequentially and/or simultaneously.
The systems provided by the invention may allow
electrophysiological measurements to be performed more quickly
and/or easily than with standard patch clamp techniques, such that
thousands of single-cell electrophysiological recordings may be
acquired in a single day. In particular, in contrast to standard
patch clamp techniques, in which a glass pipette is used to form a
high-resistance electrical seal with a biological membrane, the
systems provided by the invention preferably utilize a single,
small (e.g., several micron diameter) aperture in an at least
substantially planar substrate to provide the sealing function.
Moreover, the systems may allow cells or biological membranes to be
maneuvered to the aperture by fluid flow. Thus, these systems may
not only make the measurement easier, by reducing or eliminating
the need for a direct human operator, a microscope, and/or a
micromanipulating arm, but they also may provide a format suitable
for achieving multiple electrical seals in parallel, thereby
increasing the measurement throughput of the device.
The systems provided by the invention may be capable of forming
high-resistance electrical seals, on the order of tens of M.OMEGA.
to 1 G.OMEGA., for example, through appropriate selection and
processing of the substrate material, aperture geometry, and
attention to the way in which the biological membrane interacts
with the substrate. Preferred substrates include thin plastic
films, in which small apertures have been photomachined using a
laser. These substrates optionally may be vacuum deposited with
thin layers of glass to aid in the formation of the high-resistance
seal. Additional, suitable substrates may include silicon wafers,
in which small apertures have been produced using standard
photolithographic/wet etching techniques. In any case, individual
cells may be positioned onto isolated apertures using a suitable
positioning method, such as differential pressure.
The system and aspects thereof are described below in more detail,
including, among others, (I) single-sample measurement chambers,
(II) preferred substrate/aperture geometries, (III) high-throughput
measurement systems, (IV) channel/transporter assays, and (V)
examples.
I. Single-Sample Measurement Chambers
FIG. 2 depicts a measurement geometry with for a single measurement
chamber, in accordance with aspects of the invention. Starting with
a thin (<25 .mu.m thickness) substrate 28, a single hole 30
(.about.1 to 3 .mu.m diameter) is formed in the bottom of a chamber
32. An electrical circuit is implemented through the use of a
Ag/AgCl sensing electrode 34 in contact with an ionic saline
solution 36. A second isolated fluid chamber 38 allows fluid access
to a bottom side of hole 30 in conjunction with a bath electrode
40, thereby completing the measurement circuit. The current flowing
in the circuit is sensed by a high-impedance operational amplifier
42 and recorded by a computer controlled data acquisition system
44.
An important aspect of the invention is the ability to form a
high-resistance electrical seal 46 between the surface of substrate
28 and a biological membrane 48 without micromanipulation by a
skilled technician. To achieve this, the sample such as a cell
containing the membrane is placed in suspension in top chamber 36,
and drawn to hole 30 through the use of differential pressure
applied between bottom chamber 38 and top chamber 36. It has been
found and demonstrated that once a cell reaches a properly chosen
and engineered substrate, an electrical seal of tens of M.OMEGA. to
greater than 1 G.OMEGA. is achievable. Given this high seal
resistance level, it is then possible to isolate and measure
typical physiological whole cell currents (>50 pA) that occur
when the ion channels in the cell membrane are activated. The high
electrical resistance seal also allows for the ability to control
the voltage of the cell, a very useful feature in analyzing ion
channel activity.
To achieve voltage clamp of the membrane, an electrode must be
placed in electrical contact with the inside of the cell. This
requires electrically permeabilizing the part of the cell membrane
52 separating the two fluid chambers. This permeabilization has
been effected in the present device in two ways: (1) voltage pulses
("zapping") generated by electrodes 34 and 40; and (2) flowing
proper concentrations of antibiotics (Nystatin or Amphotericin B)
in bottom chamber 38. There also are many other types of chemicals
(e.g., gramicidin, ATP, valinomycin, etc.) that could be used to
provide electrical access to the cell interior.
II. Preferred Substrate/Aperture Geometries
The apparatus may be used with any suitable cell, organelle,
vesicle, or other membrane system. Exemplary mammalian cell lines
of interest in ion channel expression systems include Chinese
Hamster Ovary (CHO) cells and Human Embryo Kidney (HEK) cells.
These cells have mean diameters in the range of 10-20 .mu.m.
Optimum hole size in the substrate is governed by several
considerations. Holes that are too large can allow cells to pass
through the hole (as opposed to sealing) when differential pressure
is applied. In addition, holes that are too large can impede
formation of higher seal resistances. On the other hand, holes that
are too small can produce a higher electrical access resistance to
the interior of the cell once an electrical seal is formed. This
higher access resistance degrades the time resolution and voltage
control performance of the system. Given these trade-offs, a
preferred implementation features hole diameters in the range of
1-3 .mu.m, although a wider range of hole diameters (e.g., 1-10
.mu.m) is feasible depending on cell type.
Given that the preferred hole diameter is on the order of a few
micrometers, it is preferable that the unperforated substrate be
thin (e.g., <25 .mu.m), at least near the hole periphery. The
reasons for this are several. Thick substrates introduce the
problem of a very narrow pore relative to the substrate thickness,
which in turn makes it more difficult to achieve fluid access to
the membrane. Fluid contact is necessary to provide an electrical
pathway to measure ion channel currents, as well as to provide the
cell with a normal physiological environment. Also, when attempting
to gain electrical access to the interior of the cell, a long
narrow channel derived from using a thicker substrate will produce
a higher electrical access resistance than that provided by a
thinner substrate. As mentioned previously, a higher access
resistance degrades system time resolution and the ability to
voltage clamp the cell. In addition, any technique to machine the
hole in the substrate is more difficult, time consuming, and costly
when starting with a thicker substrate. As such, substrate
materials utilized in these embodiments preferably had a thickness
of less than 25 .mu.m in their entirety or at least near the
periphery of the hole.
Accordingly, an important consideration of this invention is in the
choice of the substrate used, the manner in which the substrate is
processed to form the hole and the specific geometry utilized to
make the concept workable in a high-throughput instrument. With
regards to the choice and manufacture of the substrate, two
specific embodiments of the device have been demonstrated in our
laboratory.
II.A Substrate Embodiment 1--Thin Plastic Films
In one embodiment, thin plastic films were used as a substrate. Two
types of thin films were tested, polyethylene terephthalate (PET)
(Dupont Mylar) and polyimide (Dupont Kapton), although in principle
any thin plastic film (e.g., polycarbonate, polypropylene,
polyethylene, etc.) may be used. The small 1-3 .mu.m diameter holes
then were photomachined into the plastic film using one of two
exemplary processes, although more generally any suitable process
may be used.
Holes were photomachined using a pulsed YAG laser operating at 355
nm. In this arrangement, a single laser beam drills an isolated
hole, one at a time. This beam is then scanned, typically using a
galvanometric mirror scanning system to raster scan the incident
beam over the substrate creating an array of photo-machined holes.
Such systems often employ an F-Theta lens system, which focuses as
well as redirects the scanned laser beam so that the beam remains
perpendicular to the target. The throughput of the scanning
arrangement thus is governed by the time to drill one hole and the
speed of the optical scanner. It also is possible to produce an
array of holes by scanning the film or substrate (instead of the
incident beam) and leaving the optical illumination system fixed.
Again, the throughput of this type of system is determined by the
speed of the scanning system and the time to drill a single
hole.
Holes also were photomachined using an excimer laser operating at
248 nm. In this arrangement, a photo-mask is imaged onto the
substrate, and the surface is ablated where the unmasked optical
energy is allowed to pass through to the substrate. Using a proper
mask design, the excimer imaging process can machine single or
multiple holes in the substrate simultaneously depending on the
mask configuration. Typically, a table scanning system is then used
to move the substrate to create a larger two-dimensional pattern of
photo-machined holes.
In one implementation, after the photo-machining process, the
substrates were cleaned and subjected to a physical vapor
deposition (PVD) of a silicon oxide SiO.sub.2 coating using an RF
sputtering process. The process involved pumping the system down to
.about.4.times.10.sup.-6 torr using a cryo-pump, and subsequently
backfilling the chamber with 7 mtorr of Argon. The high RF field
generated between two electrode plates then interacts with the
Argon to produce an ion bombardment of a SiO.sub.2 target. The
dislodged SiO.sub.2 then is deposited onto the thin plastic film
that is placed on a rotating platter running at 20 rpm. All
operations are run at room temperature. Coating thicknesses
implemented were in the range of 500 to 1000 angstroms.
It was determined experimentally that the SiO.sub.2 coating of the
plastic film significantly enhanced the electrical sealing
properties between the substrate and the cell membrane, increasing
the seal resistance from tens to hundreds of M.OMEGA. for the bare
plastic film to resistances on the order of 1 G.OMEGA. with the
deposited glass coating. Other implementations of the coating
process may be possible, such as using different thicknesses,
different constituents (e.g., boron doped), and different
deposition techniques (e.g., chemical vapor deposition). The
specific implementation described here should not limit the scope
of the invention.
FIG. 3 depicts two separate examples of a cell 58 positioned over a
hole 56 in a thin layer substrate 54. As shown, due to the nature
of the photomachining process, the holes are larger on one side
than the other; the diameter on the smaller side of the pore is in
the range of 1-3 .mu.m. In each of these cases, a SiO.sub.2 coating
60 is applied to the cell-side surface to improve seal formation.
Both geometries have proven to be viable in achieving good
electrical resistance between the cell membrane and the
substrate.
FIGS. 4 and 5 demonstrate typical whole-cell electrophysiological
data acquired on CHO cells transfected with the voltage gated
potassium channels Kv3.2. In this case the substrate material was
Kapton, the hole was photomachined with an excimer laser (.about.3
.mu.m diameter), and the resultant substrate was coated with a 500
angstrom SiO.sub.2 coating. The cell was positioned onto the hole
in the substrate using differential pressure of approximately 5
inches of H.sub.2O. After contacting the membrane, a seal
resistance of approximately 1.3 G.OMEGA. was measured.
FIG. 4 contains two data graphs relating to measured electrical
leak resistance between a transfected CHO cell and a SiO.sub.2
coated Kapton.RTM. polyimide film membrane pore. The top graph
represents the applied command voltage placed on the measurement
electrode. As shown, the voltage sweeps from -100 mV to +60 mV
(range of 160 mV) over approximately a 90-msec time course. The
bottom graph represents measured current after the electrical seal
was formed. As shown. the current over the same time course
increased approximately 120 pA. Since the resistance of the cell
membrane itself without ion channel activation is on the order of
10 G.OMEGA., the measured current in this example is primarily due
to leak resistance. The leak resistance, which is a measure of the
electrical seal between cell membrane and the substrate, is
computed from the data as (160 mV/120 pA)=1.3 G.OMEGA..
To demonstrate voltage control of the cell and physiological
currents, the whole-cell configuration was implemented using the
antibiotic amphotericin B to chemically permeabilize the part of
the membrane covering the hole. This was accomplished by flowing
amphotericin B at a concentration of 200 .mu.g/ml to the underneath
side of the hole. The mode of action of this compound is then to
partition into cell membranes, where it interacts with cholesterol
to form tiny channels permeable to monovalent ions. This provides a
low-resistance electrical access to the interior of the cell and in
turn allows for control of the transmembrane voltage over the
remaining unpermeabilized cell membrane.
FIG. 5 contains two data graphs relating to the physiological
measurement of the Kv3.2 channel activity after the application of
amphotericin B and under "whole cell" conditions. The top graph
represents the applied voltage sweep, which ranged from -100 mV to
+60 mV (same sweep as that of FIG. 4), providing a measure of the
voltage activity of the channel. As shown, there is practically no
current present until approximately 50 msec into the sweep
(transmembrane voltage of -10 mV), at which time the potassium
channels open and a positive current (out of the cell) is recorded.
The bottom graph represents measured current generated by channel
activity, where the voltage clamp was stepped sequentially for 90
msec intervals from a resting potential of -70 mV to the different
respective voltages labeled on the graph. As shown, for this
particular channel, current is slightly activated at a membrane
potential of -20 mV, and is greatly activated at more positive
potentials.
Although the data represented in FIGS. 4 and 5 was gathered from a
single cell on a single hole, the substrate, processing, and
experimental method utilized is entirely amenable to one where
multiple cells could be measured in a parallel architecture.
II.B Substrate Embodiment 2--Silicon Wafers
In another embodiment, standard solid-state process techniques to
produce a perforated membrane substrate. The processing started
with <100> p-type silicon wafers that had been polished on
both sides. After cleaning, a 4000 .ANG. layer of silicone oxide
(SiO.sub.2) was thermally grown on both sides of the wafer. This
layer then was followed by a 2000 .ANG. layer of silicon nitride
(Si.sub.3O.sub.4) and a second 4000 .ANG. layer of SiO.sub.2, each
of which was deposited using LPCVD on both sides. The front side of
the wafer then was patterned with photoresist to allow for the
removal of a 1 mm square section of all three oxide layers through
Reactive Ion Etching (RIE). The back side of the wafer then was
patterned to allow for the removal of a coincident 4 .mu.m diameter
section of the oxides, again through a reactive ion etch.
After stripping and cleaning, an anisotropic wet etch was performed
in EDP to produce a pyramidal shaped hole from the front side of
the wafer (1 mm square) to the oxide layers on the back side of the
wafer. This resulted in a 1-.mu.m thick, 300-.mu.m square membrane
of oxides with a 4-.mu.m diameter hole in the center. This process
may be extended to produce wafer substrates exhibiting 1 or
2-dimensional patterns of hundreds to thousands of holes.
Individual cells then were positioned onto the individual etched
holes using differential pressure, as described previously.
III. High-throughput Measurement System Description
This section describes systems for conducting electrophysiological
measurements, serially and/or simultaneously, on a plurality of
samples. These systems may include (A) a multiaperture substrate,
(B) a plenum fluidics system (C) a plenum vacuum regulation system,
(D) a sample handling/fluidics system, (E) an
electronics/measurement system, (F) an activation system, and/or
(G) a controller system, among others.
III.A System Overview
FIG. 6 shows an exemplary multiaperture system, adapted to conduct
simultaneous measurements on a plurality (e.g., an n.times.m grid)
of samples, in accordance with aspects of the invention. This
system includes a measurement platform 70 for supporting various
components of the system. These components include a plurality of
function stations, including an analysis station 72 and one or more
input stations 74, renewal stations 76, and/or cleaning stations
78, among others. These components also include a sample handling
fluidics head 80 having a plurality of dispense elements 82, an
electronics head 84 having a plurality of electrodes 86.
The multiaperture system of FIG. 6 may be controlled via any
suitable method, such as an external microcomputer 96, CRT display
98, and software user interface. The system further may incorporate
an embedded microcontroller, interfaced to the external
microcomputer, for controlling real-time functional aspects of the
instrument, including motion control, fluidics control, and
electrical data recording.
The controller further may be interfaced with a three-dimensional
mechanical gantry system 99 capable of independently moving the
fluidics head (80) and the electronics head (84). The fluidics and
electronics heads may, without loss of function, independently
comprise single probes, n.times.1 (1-dimensional) probes, as shown
here, or n.times.m (2-dimensional) probes. Thus, the combination of
the controller and gantry systems allows for the spatially
selectable transfer of potential drug candidates to the various
n.times.m "wells" of the multi-well measurement substrate using the
fluidics head, the spatially selectable activation of caged
compounds, and/or the spatially selectable electrical recording
from samples using the electronics head.
The system components, most generally, be configured for
independent and/or coordinated movement, with the individual
components (or portions thereof) moveable and/or fixed, as desired,
consistent with an ability to bring components into registration or
alignment as needed for particular functions. For example, the
fluidics head and a sample holder may be brought into register by
moving the fluidics head, the sample holder, or both, using any
suitable registration device or mechanism.
III.B Measurement Platform
The measurement platform generally comprises any mechanism such as
a planar surface for supporting and/or maintaining the spatial
arrangement between some or all of the components of the
measurement system.
FIG. 7 is a top side view of one implementation of the measurement
platform for use in high-throughput electrophysiological
measurements, in accordance with aspects of the invention. This
layout includes eight separate linearly disposed fixture positions
124-131. The electrical sensing head (104) can access three
positions (124-126), the multi-channel dispensing head (106) can
access six positions (126-131), and both heads can access one
position (126). More generally, the system platform layout may
include any suitable number of positions, for any suitable
functions, disposed and accessed in any suitable manner.
Position 126 is an analysis station, referred to as the plenum,
which in turn is used to support the individual measurement
substrates containing samples. This position may be accessed by the
multi-channel dispensing head, to dispense samples, screening
compounds, and the like, and by the electrical sensing head, to
make electrophysiological measurements. The plenum system reservoir
creates an air-tight seal by locking the measurement substrate into
position atop the plenum using a vacuum-induced differential
pressure between the measurement substrate carrier and an o-ring
situated on the plenum. This air-tight seal allows fluid in the
plenum reservoir to be maintained at slightly less than atmospheric
pressure, thereby introducing a differential pressure across the
membrane that forces fluid from the top chamber through the
individual apertures into the common lower reservoir. The resulting
flow pulls individual suspended cells (or cell membranes) in
multi-well compartments down onto the individual apertures in
parallel, without direct human intervention. In addition, once the
cells contact the membrane surface, the continued use of
differential pressure enhances the formation of high-resistance
electrical seals between the substrate material and the cell
membrane.
Positions 128 and 131 are input stations, from which potential
biological screening compounds may be obtained. The footprint for
these positions preferably is compatible with 96, 384, and/or
1536-well microplates, as these are common receptacles for
potential drug candidates (agonists or antagonists) used by the
pharmaceutical industry.
Position 130 is another input station, from which extracellular
fluid may be obtained. This fluid preferably comprises a
physiological saline solution for transfer by the fluidics head to
the top side of the measurement substrate at position 126. The
station may include a removable boat that holds the saline
solution; alternatively, or in addition, the station may be
automated by priming it for automatic fill and drain via a
peristaltic pump and a vacuum-assisted waste bottle.
Position 129 is yet another input station, from which cells or
other biological samples may be obtained. This station also may
include a removable boat that contains cells or other user-prepared
biological material in suspension for transfer by the multi-channel
fluidics head to the top side of the measurement substrate.
Cellular samples may be maintained as a slurry, for example, with
cell densities on the order of about 10.sup.6 cells/ml. The volume
of cell slurry required for an experiment depends on the number of
wells used in the experiment and on the volume of fluid dispensed
into each well; for 384 wells, with 3-4 .mu.l dispenses, the volume
of slurry required is less than about 1.5 ml.
Position 124 is a renewal station for replenishing the chloride
coating of the electrodes. This station may include a removable
boat that contains a solution (commonly bleach) for depositing
chloride on the sensing pins of the multi-channel electrical read
head. More generally, the station may include any apparatus or
material suitable for maintaining, replenishing, and/or
rejuvenating the electrodes.
Positions 125 and 127 are cleaning or wash stations for the
electrical head and the multi-channel fluidics head, respectively.
The electrical and fluidics heads should be cleaned whenever they
come into contact with potentially biologically active test
compounds, to reduce or prevent carryover that may affect future
measurements. The two cleaning stations each include a manifold of
input ports that preferably matches the dimensionality of the
associated electronics or fluidics head, or a portion thereof
(here, both 12 channels). The two stations employ a design whereby
cleaning fluid is pumped using a peristaltic pump from a source
bottle through individual access ports 134 to overflow into a
respective catch basins 136. The stations may use any suitable
cleaning solution to clean both the sensing pins from the
electrical head and the dispense elements of the multi-channel
fluidics head, for example, water and a cleaning solvent such as
10% ethanol for the fluidics head and a saline solution for the
electronics head. The inside of the needles from the fluidics head
may be washed by performing fast aspirate/dispense cycles in
association with flowing fluid through the individual input ports
134. Compartmentalizing the individual wash ports reduces wash
volume and the potential for well-to-well contamination. It also
forces fluid around the outside of the individual dispense needles
of the fluidics head. The waste basins drains directly into a
vacuum-assisted waste bottle. The close proximity of the wash
stations (125 and 127) to the analysis chamber (126) reduces
overall assay time by reducing the distance the respective
electronics head (104) and fluidics head (106) must travel in
performing repetitive and time-consuming washing steps during
biological assay protocols.
The input fluids typically include two saline solutions, at least
for general electrophysiological experiments. The first saline
solution (source 136) comprises a mixture of salts that mimics the
internal cytoplasm of a living cell e.g., containing high
potassium. This solution (denoted "internal:" buffer) may be used
on the bottom side of the plenum fixture 154, which is the side by
which electrical access to the interior of the cell is achieved.
This solution is analogous to the fluid inside the pipette in
classical electrophysiology, and may be pumped in and out of the
plenum system by a peristaltic pump 140. The second saline solution
(source 142) comprises a mixture of salts that mimics the
extracellular solution, e.g., containing low concentrations of
potassium. This solution (denoted "external" buffer) may be used on
the top side of the measurement substrate, and may be added by the
multi-channel fluidics head to the separate wells of the
measurement substrate by accessing at position 162 and dispensing
into the top side of the multi-well carrier at position 154. In
operation, the constituents of both the internal and external
saline solutions may vary greatly, as is common in classical
electrophysiology.
The input fluids also typically include a perforation solution
(source 144), which can be accessed by the plenum pump 140 through
proper valve actuation. This causes the perforation solution to
flow into contact with the bottom side of the membrane substrate
and thus to the biological membrane. Through chemical permeation,
this solution serves to provide a low-resistance electrical pathway
to the interior of the cell membrane. The perforation solution
preferably comprises an "internal" (high potassium) saline
solution, mixed with an appropriate concentration of a chemical
that subsequently provides a low electrical resistance access to
the cell. This chemical may include, among others, amphotericin B,
nystatin, gramicidin D, paradaxin, ATP, and so forth. The system
may replace the initial plenum solution 136 with this new solution
144 using the plenum peristaltic pump 140. Preferably, the fluid in
the lower chamber of the plenum may be exchanged without
introducing significant pressure pulsation or static pressure
changes that could disrupt the process of high-resistance seal
formation between the biological membrane and the multi-well
substrate. During plenum fluid exchange, or at the end of the
experiment, the expelled plenum solution is pumped out to a
separate waste container 166 using pump 140.
This example shows only two plenum input solutions 136, 144;
however, in practice, it is possible to include as many input
solutions as necessary or desired. The ability to exchange multiple
internal solutions, i.e., the solution that has access to the
inside of the cell, is not available in classical electrophysiology
using a standard pipette.
FIG. 9 shows the location of input 136 and output 168 reservoirs,
relative to an instrument housing. Here, without limitation, to
facilitate use, the input fluids are located on the left side of
the instrument, and the output (waste) fluids are located on the
right side of the instrument layout. The system as shown includes
four fluid inputs and two fluid outputs, but more generally may
include as few or as many of each as necessary or desired.
III.C Multi-Well Substrate and Carrier
The multiaperture substrate generally comprises any mechanism
having a plurality of holes or apertures about which a
corresponding plurality of samples may be positioned and/or sealed
for analysis. The substrate preferably has one aperture per sample
well, although in some configurations there may be two or more
apertures per sample. The substrate also preferably allows each
sample to be independently exposed to reagents, candidates, and/or
other materials, for example, via separate sample wells in fluid
isolation from other sample wells, at least on one side of the
substrate.
FIG. 10 shows an exemplary multiaperture substrate, comprising a
multi-well membrane carrier 350, a thin membrane substrate 352, and
the receiving plenum fixture 354. The carrier 350 comprises the
top, well-containing portion of the multiaperture substrate. The
carrier may be formed of any suitable material, by any suitable
process, for example, injection-molded polystyrene. The membrane
substrate 352 comprises the surface and associated apertures onto
which samples are sealed for analysis. This substrate also may be
formed of any suitable material, by any suitable process.
Typically, the substrate is formed of a thin plastic film (such as
a Kapton.RTM. polyimide film or a Mylar.RTM. polyester film) that
has been photo-machined (or otherwise provided) with an array of
single apertures that match the geometry of the carrier, i.e., one
aperture per well. The carrier and cleaned, machined membrane
substrate may be joined using any suitable mechanism (e.g., by a
non-toxic adhesive or ultrasonic bond), forming an electrically
isolated fluid chamber 356 on top of each aperture in the membrane.
For example, this bonding may be achieved by applying a layer of
adhesive between the membrane and carrier, which then is cured
through a combination of time, heat, and/or ultraviolet light. The
assembly of carrier and membrane then forms a single substrate
assembly that is assembled and packaged, preferably in a
hermetically sealed pouch, in a clean room. Clean-room techniques
are advantageous to reduce or eliminate foreign debris that may
otherwise be introduced into the individual wells and potentially
plug the small aperture at the bottom of each well during initial
fluid flow.
The exemplary system described here includes a rectangular grid of
48.times.8 apertures that forms a 384-well substrate. The apertures
have a 2.25-mm center-to-center spacing, and the well volume that
is formed by the carrier and membrane holds approximately 15 .mu.l.
This geometry is only one of many that could be implemented in
building such a device. For example, the illustrated design could
be extrapolated to form an array of 48.times.32 apertures (1536)
wells having a 2.25-mm spacing. The choice of center-to-center well
spacing ideally should conform to a standard microplate format, so
that the multi-channel fluidics head readily can access both
compound plates and the membrane carrier. The industry standard
microplates have 96, 384, and 1536 wells with 9, 4.5, and 2.25 mm
center-to-center well spacings, respectively.
As shown in FIG. 10, upon use, the multi-well assembly is lowered
into the top access of the plenum 354, where it may be clamped via
a vacuum port 358 and o-ring assembly 360 located on the top
surface of the plenum. The seal achieved between the o-ring and the
outer rim of the membrane carrier isolates the internal chamber of
the plenum. This, in turn, allows regulation and alteration of the
internal plenum fluidics path at pressures slightly below
atmosphere, as just described. The use of a vacuum chuck
arrangement is a convenient (fast and efficient) interface for
loading membrane/carrier substrates into the plenum fixture.
III.D Plenum System
The plenum system generally comprises any mechanism for adding,
removing, and/or replacing fluids and associated materials from the
bottom side of one or more sample wells, sequentially and/or
simultaneously, while typically providing a means to control the
differential pressure across the substrate. The plenum system
preferably is closed, so that a differential pressure can be
introduced, controlled, and/or regulated between the top side of
the measurement substrate, which is at atmospheric pressure, and
the bottom side of the measurement substrate, which generally is
held at a slight vacuum. This differential pressure may be used, as
described above, to position a cell, vesicle, and/or other sample
that is in the fluid on the top side of the membrane onto the small
pore(s) located in each respective well of the measurement
substrate. In addition, by controlling the differential pressure
very precisely, and with the appropriate timing, this pressure can
be used to facilitate formation of a high resistance electrical
seal between the biological membrane and the measurement
substrate.
The plenum system also may be used to replace air that initially is
in the system with fluids from one or more fluid inputs. This
replacement is necessary to provide a continuous fluid path, which
allows a complete electrical circuit to be formed between the
solutions above and below the membrane. To accomplish this task,
air must be removed from the various fluid pathway lines, as well
as from the small aperture in the measurement substrate. Removing
the air from the substrate is a difficult proposition, because the
microscopic geometry of the aperture typically is a long narrow
channel.
The plenum system also may be used to replace one fluid in the
system with another during the measurement process, after
electrical seals have formed. To do this effectively, without
unduly disrupting the seal, the plenum system should be able to
reduce or minimize pressure perturbations, as well as to control
the differential pressure during the exchange process. These and
other aspects of the plenum system are described below, including
(1) the plenum fluidics subsystem, and (2) the plenum vacuum
regulation subsystem.
III.D.1 Plenum Fluidics System
FIG. 11 is a schematic representation of an exemplary plenum
fluidics system, in accordance with aspects of the invention. This
system generally comprises any mechanism for regulating fluid
access to the bottom side of the measurement substrate. The system
includes a plurality of fluid pathways (solid lines 171),
preferably constructed of flexible silicone tubing and pneumatic
pinch valves. This arrangement allows for a stable, biologically
inert pathway, which, with proper maintenance, can be kept clean,
since the fluid is confined to the silicone tubing and not exposed
to other valve components. The pumping action may be provided by
any suitable mechanism, such as a dual, 4-roller peristaltic pump
174. This allows for the maximum pumping efficiency, while
minimizing the "pulsing" of the fluid flow as the rotor turns over
the tubing.
The system also includes two debubblers 176 and 178, positioned on
opposite sides of the plenum (169). These debubblers have several
functions. First, and foremost, they are a convenient way to remove
macroscopic bubbles from the system. Specifically, as a bubble
floats into the partially filled debubbler tube, the bubble will
float to the top and be removed from the fluid path. Second, the
debubblers also serve as convenient control points for vacuum
control. Third, the debubblers also act as a capacitive reduction
to kinetic perturbations in pressure introduced by the pump during
fluid flow.
The system may be filled with fluid by an appropriately sequenced
actuation of various valves, in combination with pump flow. To
remove bubbles in the system that cling to the tubing or the plenum
itself, a "pulsing" procedure may be implemented. This procedure
may involve (1) running the pump at a moderate speed, (2)
initiating an increase in positive pressure in the plenum by
closing off the output side of the plenum at valves 180 182 and
184, and (3) relieving the increase in positive pressure by opening
valve 180 temporarily. The resulting pressure pulse may clear
bubbles out of the system, which fluid flow via the peristaltic
pump alone typically is not sufficient to do. This debubbling is
performed prior to adding cells or other samples to the system,
since the pressure perturbations induced during this "priming"
procedure may be too great to maintain an electrical seal between
cell and membrane. The pulsing may be performed while maintaining a
negative absolute differential pressure in the system, even during
the pulses, ensuring flow from the top side to the bottom side, so
that potentially recycled and "dirty" solution from the bottom side
of the plate should not flow backwards to the topside, thereby
plugging the hole.
The fluid in the system may be exchanged with other fluids, also by
an appropriate use of various valves and pump flow. To exchange
fluids, the plenum system may be opened at an input point, to
accept new fluid, and at an exit point, to expel current fluid. For
example, to replace fluid from a bottle 170, which previously has
been added to the plenum, with fluid from another bottle 172, a
valve 186 may be opened, and a 3-way valve 188 may be switched from
the closed-loop flow position to the open-to-waste position. To
reduce or eliminate pressure perturbations, and to control
differential pressure during the "exchange" procedure, it often is
useful to control the vacuum pressure in the waste bottle 190 and
to maintain it a proper relative height to the other control points
in the system.
III.D.2 Plenum Vacuum Regulation System
FIG. 12 is a schematic representation of an exemplary plenum vacuum
regulation system. The plenum vacuum regulation system generally
comprises any mechanism for controlling (e.g., maintaining,
regulating, and/or monitoring) the differential pressure across the
measurement substrate 192. This differential pressure generally may
assume any value consistent with its intended function(s), which
may include facilitating sealing of samples across the aperture
and/or disrupting or destroying the portion of the sample sealed
across the aperture. The differential pressure for proper
high-resistance seal formation typically assumes values in a range
between 0 to 10 inches of water of vacuum with respect to
atmosphere, with a precision of about 0.05 inches of water. This
vacuum range encompasses the range that typically is applied to the
pipette during seal formation in classical electrophysiology.
The plenum vacuum regulation system may include, among others, one
or more of each of the following components: (1) a vacuum (or
pressure) sensor, for sensing pressure, (2) a vacuum (or pressure)
regulator, for controlling vacuum, (3) a debubbler(s), for reducing
or eliminating bubbles, (4) a pump, for applying pressure, (5) a
line, for routing fluid between sources, sinks, the plenum, and/or
other positions, and (6) an environmental sensor and/or controller,
for sensing and/or controlling environmental conditions, such as
temperature, pH, and the like.
The exemplary regulation system includes an electronically
voltage-controlled vacuum regulator 194 for controlling the
differential pressure in the plenum in the desired range. A
regulated vacuum output line 196 responds to a vacuum sensing line
198 to maintain a desired vacuum level, as set by an analog voltage
control. Both the regulated output line and the sense line are
connected to a trap bottle 200, which serves as a protection
mechanism for the sensor, as well as a form of ballast to aid the
dynamics of the control system. The outputs of the regulator are
fed to two debubblers 202 and 204 (as referenced in FIG. 11) in the
plenum system, and to a plenum waste bottle 208, to maintain proper
vacuum control points throughout the plenum system.
Control of the vacuum regulation system is made difficult by the
small desired control range, typically 0 to 10 inches of water
vacuum, relative to atmosphere. To facilitate control, the
pre-debubbler 202 control point is located approximately 3 inches
above the plenum interface, thereby providing 3 inches of water
positive pressure offset (when the system is primed and filled with
fluid) between the debubbler control point and the plenum 206. This
provides a convenient control offset, such that the regulator works
in a stable, linear range during normal operation at about 3 inches
H.sub.2O of vacuum. This, in turn, allows the regulator system to
control the differential pressure in the plenum accurately all the
way to zero differential pressure relative to atmosphere.
The regulation system also may include a gauge or sensor 208, such
as an analog gauge, that measures, in real time, the pressure
differential between atmospheric pressure and the plenum. This
gauge may be used for feedback to the vacuum regulator or as a
quality control system monitor to indicate to the user that all
systems are operational and functioning properly. For example, if
the membrane substrate is not sealed properly to the plenum,
thereby allowing air into the system, the gauge will provide a
mechanism for determining that the system is not at the proper
differential pressure and so can be used to warn the user of a
potential problem.
III.E Sample-Handling Fluidics Head
The sample-handling/fluidics system generally comprises any
mechanism for adding, removing, replacing, and/or transferring
fluids including samples, reagents, and/or drug candidates to the
top side of one or more sample wells, sequentially and/or
simultaneously.
The sample handling fluidics system is configured to introduce
materials independently into the respective wells of the
measurement substrate, as desirable in an instrument designed for
parallel simultaneous measurements. These materials may include,
among others, (1) physiological saline buffer, (2) suspended cells,
cell membranes, vesicles, or beads with adherent membranes, and/or
(3) experimental chemical entities, for example, for the purpose of
analyzing their effect on the electrophysiology of the biological
membrane. The measurement fluidics system may obtain fluid from a
source reservoir or multiwell plate and then dispense the same
fluid in a destination reservoir, e.g., the multi-well carrier,
using one or more pipette channels. Once cells are added to each
well, "cell positioning" may be accomplished using any suitable
method, for example, by applying differential pressure across the
substrate to increase fluid flow through each aperture, as
described earlier. The cells then are carried by the fluid flow to
the single aperture in each well of the multi-well chamber, at
which time an electrical seal can form.
The accuracy, precision, and volume specifications of the fluidics
head may be selected as necessary or desired, depending on the
types of samples under study, the intended throughput of the
instrument, and the intended quality of the measurements. In a
preferred embodiment, the fluidics head is capable of accurate
(.about.2%) and precise (<2% CV) fluid aspiration and dispense
cycles, at volumes of about 3 to 4 .mu.l. The ability to dispense
fluids with accuracy and precision is motivated by the desirability
of adding known concentrations of given biologically active
compounds to the sample compartments to facilitate repeatable,
comparative biological assays, as well as to provide for
well-to-well comparison of compound activity. The ability to
dispense low volumes of fluid is motivated or necessitated by the
geometry of the membrane carrier, which in the preferred embodiment
has a full-well capacity of about 15 .mu.l, and by the functional
requirements of the assay, which may involve making 3-4 additions
per assay.
FIG. 11 shows an exemplary fluidics head 212, in accordance with
aspects of the invention. This head includes twelve (12) dispensing
elements 214 and an "extra" cherry-picking dispense element 216 off
to the side. Here, the dispense elements comprise stainless steel
needles, coated both inside and out with Teflon.RTM.
tetrafluoroethylene polymer to reduce the effects of compounds
adhering to the surface of the needle and causing a carry-over
problem for subsequent runs. Fluid transfer (e.g., the process of
loading and/or-dispensing fluids) may be accomplished using any
suitable mechanism, including contact and/or noncontact dispensing.
Fluid transfer preferably is accomplished using a piston/o-ring
manifold assembly 218, driven by a micro-stepping motor and
precision lead screw assembly, that deposits fluid in individual
sample compartments 220. This motor and other stepping motors in
the system are disabled before making electrical measurements, to
reduce noise.
The "extra" dispense element in this design covers cases in which
it is necessary or desirable for the instrument to perform single
channel head pipetting. For example, in the screening mode, not all
individual wells of the membrane substrate will form high
resistance electrical seals and provide physiologically relevant
data. Moreover, individual cells or membranes may vary in their ion
channel expression levels, so that not all of the biological
samples will contain the ion channel of interest.
If the compounds are added to the measurement substrate quickly
using a multi-channel pipettor, without regard to which wells are
physiologically viable, then it is likely that some of the test
compounds will not reach a physiologically viable well. The
probability of such an event depends on the probabilities of the
cell forming a high-resistance seal in each individual well, the
cell and other components of the system allowing electrical access
to the cell to clamp the voltage, the cell having the ion
channel(s) of interest, and the amount of replication used in
adding the test compounds, among other factors.
One approach that may increase the probability of obtaining a valid
test sample is to pre-sample the wells, and then only to add test
compounds to those wells that are known to be physiologically
viable. The drawback of this approach is that it requires extensive
pre-testing and a lot of single channel pipetting, which may
greatly increase the time necessary to sample the entire multi-well
substrate.
Another approach that may increase the probability of obtaining a
valid test sample is to add the test compounds from a multi-channel
pipettor, without regard to which wells have adequate electrical
seals and physiological currents. In particular, given a reasonable
percentage of well hits, the most expedient way to cover the test
wells is to add the compounds systematically to "most" of the plate
using the multi-channel pipettor (in duplicate or triplicate
perhaps) and then to go back after the experiment with a single
channel pipettor to "re-test" (after the fact) those compounds that
did not have a viable data point. This type of retest strategy
relies on using a single channel pipettor during the "retest" phase
so as not to corrupt neighboring test wells or to waste reagents
and to use the remaining "good" wells efficiently, while minimizing
the amount of single-channel pipetting that must occur.
The use of the fluidics head may be illustrated by an example.
Suppose that all compounds from a 96-well drug plate were added in
triplicate to 288 wells (3.times.96) of a 384 well membrane
substrate. This operation could be performed quite quickly using a
multi-channel pipettor. Now, further suppose that physiological
currents were measured in 50% of the wells, i.e., that there were
192 "good" wells with high resistance electrical seals and
measurable physiological currents. If this success rate were
distributed randomly, we would then expect 1/8 (12) of the
compounds to have 3 good measurements, 3/8 (36) of the wells to
have 2 good measurement, 3/8 (36) of the wells to have 1 good
measurement, and 1/8 (12) of the well have 0 good measurements. In
addition, there would be 96 (i.e., 384-288) virgin (i.e., unused)
wells left to be tested, which at a 50% success rate would leave 48
available wells for further testing. The strategy then would be to
go back and pick up each of the 12 compounds that yielded 0 valid
test points on the first pass and to add them systematically to
tested viable wells one at a time. This could be done as
singletons, duplicates, triplicates, or the like. If desired and
timely, this procedure also could continue on to the compounds that
have only one good measurement until all the "good" wells were
utilized. The optimum strategy, of course, depends on the hit rate
(here assumed 50%) and the amount of time allotted for the
assay.
The problem with this approach is that it requires two separate
fluidics heads: one multi-channel and the other single-channel.
This necessitates using either two sets of four-axis motion
controllers (X, Y, Z, dispense), one for each head, or one
four-axis motion controller, with separate, individually docked
heads. The fluidics head described here may overcome both of these
complexities, by attaching a separate dispense element to the
assembly, offset in X, Y, and Z (height). In multi-channel
operation 222, this extra dispense element clears the other
fluidics positions (due to the X Y Z offset). Conversely, in
single-channel operation 224, the multi-channel head does not
interfere with anything on the platform, and the single tip can
access any position of the measurement substrate or compound plates
(due to the clearance provided by the Z offset). The advantage of
this approach is its simplicity in that it avoids having a
multiplicity of motion control and/or docking systems. This
approach and the preferred design are extendable to other
one-dimensional or two-dimensional formats, including lines and
grids.
III.F Electronics Measurement System
The electronics/measurement system generally comprises any
mechanism for applying and/or measuring electrical potentials
and/or currents from one or more samples, in one or more sample
wells, sequentially and/or simultaneously.
FIGS. 14 and 15 show an exemplary electronics/measurement system,
in accordance with aspects of the invention. Electrophysiological
measurements may be performed on cells, using this system, by
forming an electrical circuit across each individual aperture in
the substrate. This may be accomplished using suitable electrodes,
positioned on opposite sides of the membrane, for example, a sense
electrode positioned above the membrane, and a ground electrode
positioned below. For convenience, particularly in multichannel
embodiments, electrodes may be mounted and manipulated collectively
using an electronics head. The electronics head may include a
plurality of individual measurement probes, each capable of
functioning as a sensing (or ground) electrode for an individual
well of the measurement substrate, sequentially and/or
simultaneously. These electrodes may be organized as an array
(e.g., with m.times.n elements, where m, n=1, 2, 3, . . . ),
preferably corresponding to the spacing of the measurement wells,
or a multiple thereof, in at least a portion of the measurement
substrate. The electronics head may be capable of two- or
three-dimensional motion, enabling it to move between the various
wells of the measurement substrate, as well as to a wash station,
where the individual sensing electrodes can be washed between
experimental runs. Each sensing electrode may be tied to its own
high-impedance amplifier arrangement, consistent with that
necessary for such measurements, preferably located in the housing
of the electronics head. The analog output signals for each of the
respective output amplifiers may be digitized by appropriate
analog-to-digital (A/D) converters and transferred to an internal
(onboard) computer and/or an external computer for further
processing.
The individual circuits may be completed by the addition of a
suitable electrolyte (e.g., saline) solution in each individual
well of the measurement substrate above the membrane and by the
introduction of saline solution below the membrane via a plenum, as
described above. A common ground electrode may be located in the
plenum fluid reservoir, thereby completing the measurement
circuit.
FIG. 14 shows a partial schematic of an exemplary electronics head
226, in accordance with aspects of the invention. This exemplary
electronics head is configured, consistent with the discussion
above, to move a plurality of sensing electrodes 228 to the various
wells 230 of the multi-well measurement substrate 232, thereby
completing a separate electrical circuit 234 for each of the
measurement wells. The electronics head includes several
measurement "pins" 228. Each pin typically is a silver or
silver-coated wire. In recent implementations, the pins have
comprised silver-plated stainless steel to improve their rigidity.
Any type of wire material that can be silver-plated could
potentially suffice. This wire, in turn, is treated, typically with
a hypochlorous acid, to give it a silver/silver chloride coating.
The current-carrying mechanism between the biological cell or
membrane and the pin then is transferred via a saline solution
containing chloride ions, which react with the silver/silver
chloride composite of each pin. The advantage of using the
silver/silver chloride electrode is that it has an extremely low
junction potential.
Once on the pin, this current may be converted to a voltage via a
high-gain, low-noise trans-impedance amplifier 236. The voltage
signal then is sent off to a multi-channel analog-to-digital A/D
converter 246, which digitizes the voltage and saves it as a
digital value in computer memory. Each electrode pin has its own
channel; however, for the sake of simplicity, not all are shown in
FIG. 14.
The ground side of each measurement circuit may be accomplished via
a suitable electrode, such as a silver/silver chloride pellet 238
that is located in the bath solution 240 in the plenum. The bath
solution in this embodiment should contain chloride ions for the
silver/silver chloride electrode to function properly. Once the
membrane carrier is sealed onto the plenum 242, for example, via
the o-ring assembly 244, as previously described, the external
saline solution may be introduced to each individual well (on the
top side of the membrane carrier), and the internal saline solution
may be introduced to the plenum (on the bottom side of the membrane
carrier) via the main plenum channel, thereby completing the
circuit.
The preferred functionality required of the electronics head is to
make current measurements for each measurement well, as well as to
maintain or alter the voltage across each individual well and
therefore across the biological membrane. To be useful for
measuring typical ion channel currents, this amplifier arrangement
should be capable of clamping the voltage over physiological
voltage ranges (at least about -100 to 100 mV), as well as be able
to detect currents on the order of 10.sup.-12 Amps with a temporal
bandwidth of about 10 kHz. These specifications are rather
demanding, but may be attained using state-of-the-art operational
amplifier circuits and printed circuit board layouts. In addition,
depending on the number of measurements to be made in parallel,
these specifications can place demanding requirements on the
multi-channel A/D converter.
In some cases, it is necessary to maintain or hold the voltage
across the biological membranes for a fixed period of time before
actually making measurements. For example, many ion channels of
interest require a "set-up" time at particular voltages to effect
different conformational states of the channel prior to
measurement. In some extreme cases, this "set-up" time may be as
long as several minutes. This can have a detrimental impact on
measurement throughput in cases in which the number of "wells" to
be measured greatly exceeds the number of sensing electrodes.
Since it is much simpler to hold and maintain the voltage in a
given measurement circuit, than it is to be able to actively change
the voltage and in-turn digitize high-fidelity current
measurements, a multiplexing scheme becomes attractive. In this
geometry, some electrode pins are used to hold and maintain the
voltage level across the membrane, while other sensing pins are
using to make the high-bandwidth current measurements. When the
functional aspects of the assay dictate, the roles of the "sensing
pins" and the "holding pins" can be reversed thereby increasing the
functionality of the device.
FIG. 15 shows an example of such a system, whereby two separate
banks of electrode pins are utilized. A digital control line 248
controls the voltage input to two banks (250 and 252) of sensing
pins via the selectable input lines 251 and 253. These input lines
are connected directly to the non-inverting input of a low-noise
operational amplifier. One control line 254 contains a voltage
waveform going to the "active" sensing bank. The other input
control line 256 is maintained at a fixed holding level. The
respective outputs of the two amplifier banks then are multiplexed
to a multi-channel A/D converter 258 via a multi-channel analog
multiplexer 260 and digital multiplexer control line 262.
When amplifier "Bank 1" (250) is active, a time-varying voltage
waveform can be input to the bank, and the output of the bank can
be sent to be digitized via the A/D converter. At the same time,
"Bank 2" (252) is maintained at a fixed voltage, and the outputs
are disconnected from the A/D converter. These pins thus would be
used to maintain a holding potential across the biological membrane
during a pre-measurement set-up time. Upon initiation by the
digital control lines, the two roles of the amplifier banks can
then be reversed.
This type of multiplexing scheme has many advantages for
high-throughput electrophysiological measurements. The ability to
voltage clamp many wells simultaneously at the holding potential
greatly increases throughput for assays requiring significant
holding times. As an example, many sodium channels require 30 to 60
seconds of hold time at a negative voltage (e.g., -90 mV) before
they will respond to a voltage stimulus. The ability to hold many
samples simultaneously, and to multiplex the sensing electrodes
between "output hold potentials" and "variable output/input sensing
potentials," essentially removes this "inactivation state" in many
wells in parallel, thereby improving the overall throughput of the
measurements. The alternative would be sequential hold times and/or
the necessity to move the electronics head between measurement
locations.
In addition, electrically switching between amplifier banks is much
faster than physically moving a single bank to a new set of
measurement wells, also increasing throughput. Multiplexing the
outputs at the electronics head itself, rather than at the A/D
converter, which may be located some distance away, greatly
simplifies the number of conductors that must be routed to the A/D
converter. Lastly, this architecture is directly extendable to
large numbers of electrodes, without making impractical demands on
the number of channels and throughput of the A/D converter.
The electronics system described in this section more generally may
be implemented for any suitable number and combination of amplifier
banks (holding and active) and individual pin-counts (i.e., 12, 24,
48 etc.). The system may, for example, be used with a 1536-well
measurement system and a 192-pin electronics head, among others,
multiplexed to a 12, 16, or 48 channel A/D converter. This
exemplary system would allow the voltage to be held on up to 192
wells, at the same time.
III.G Activation System
The activation system generally comprises any mechanism for rapidly
activating (and/or deactivating) effector compounds in one or more
sample wells, sequentially and/or simultaneously. For example, the
system may use suitable light from a suitable light source to
activate a photoactivatable compound, and/or a suitable voltage or
change in voltage from a suitable voltage source to activate a
voltage-activated compound, and so on. The system preferably uses
light to "uncage" a photoactivatable "caged compound" comprising a
ligand, a candidate ligand modulator, and/or the like.
FIG. 16 shows an exemplary photoactivation system, in accordance
with aspects of the invention. This system, which is associated
here with an electronics head 282, includes a light source module
290 for generating light and a light coupler 292 for directing that
light to one or more sample wells in a measurement substrate. The
photoactivation system and the electronics/measurement system are
adapted to work in concert to concurrently photoactivate compounds
and record electronic signals such as voltages and/or currents from
the same wells. This adaptation allows for the rapid and direct
activation of effector compounds (e.g., through UV flash photolysis
of a caged compound) and the simultaneous electrical recording of
time-critical, ligand-activated ion channel or ion transporter
events.
The light source module generally includes a light source 294
capable of generating light that in turn is capable of or adaptable
to activate the photoactivatable compound. Suitable light sources
may include continuous and/or time-varying sources, such as arc
lamps, flash lamps, lasers, photodiodes, light-emitting diodes
(LEDs), and/or electroluminescent lamps, among others. Preferred
light sources for activating caged compounds include ultraviolet
(UV) light sources, such as UV lasers and UV lamps. The light
source module (and/or other components of the system) may control
or modify one or more properties of the light outputted by the
light source, such as its wavelength, intensity, polarization,
and/or the like (e.g., using spectral filters, intensity filters,
polarizers, and/or the like, respectively). The light source module
(and/or other components of the system) also may control the timing
of the delivery of the light onto the sample, including the start
time and the duration of the illumination. This control may be
achieved by pulsing the light source and/or by adding intervening
gating optics, such as filters, shutters, acousto-optic modulators,
and so on.
The light coupler generally comprises any mechanism for directing
light from the light source onto one or more of the samples.
Suitable light couplers may include optical fibers 298, free space
optics, and/or evanescent wave coupling through the base of the
substrate. Suitable light couplers further may include conventional
optical elements such as mirrors, beam splitters, diffusers,
collimators, telescopic optics, and/or the like, which may be used
as appropriate in place of, or in addition to, the components
previously described. The light may be directed onto the same well
or sets of wells in contact with the electrical system, or a subset
thereof, to facilitate coordinated activation and electrical
measurement. For example, in FIG. 16, there is a one-to-one
correspondence between electrodes and illuminable wells.
The photoactivation system may be controlled by a central
processing unit (CPU). The CPU preferably is capable of controlling
the optical pulse width and intensity of the source, so that the
timing, duration, and light energy of the ultraviolet exposure can
be controlled automatically.
III.H Exemplary Experimental Protocols
This section describes exemplary experimental protocols for using
an electrophysiological measurement apparatus, in accordance with
aspects of the invention.
The protocols begin with flushing and rinsing the instrument with
the appropriate saline solutions, after which the user loads a
multi-well carrier into the plenum fixture, which then is sealed by
the application of high vacuum. This creates an air-tight vacuum
controlled reservoir system on the bottom side of the membrane. The
fluidics head then aspirates enough volume from the external
(low-potassium) buffer reservoir to dispense approximately 3.5
.mu.l into every well of the 384 (8.times.48) multi-well carrier.
Once this is accomplished, the plenum fluidics system begins
pumping high potassium (internal) saline solution into the plenum
fluidics system. Using a combination of fluid flow and pressure
pulsation, the plenum replaces the air in the plenum system
reservoir with the high potassium solution. This procedure is
carried out while maintaining a slight vacuum (.about.7-9 inches of
H.sub.2O) with respect to atmosphere in the plenum system, ensuring
that any fluid flow across the measurement substrate is from the
topside to the bottom side.
The fluidics head optionally may be set to dispense fluid into a
user-selectable subset of the sample wells, such as one-fourth or
one-half of the wells, among others. The partially filled plate
then may be used for experiments, such as assay development, in
which fewer (e.g., 96 or 192) measurements are necessary. The
remaining, pristine portion of the plate then may be used for a
subsequent assay, if at all.
Once the system is primed, the electronics head will sample each
well of the multi-well substrate electrically to test for
electrical continuity through the from the top side, through the
photo-machined hole, to the bottom side ground electrode on each
well of the substrate. This operation is reminiscent of the "bath
test" in classical electrophysiology. Under normal operation, and
with typical saline solutions, the equivalent resistance of each
photo-machined hole is on the order of 2 to 4 MOhms, although other
holes sizes and effective resistances could yield acceptable
results. After the plate is primed, during the "hole test"
measurement, the differential pressure across the measurement
substrate is turned off, so as not to pull debris through the open
hole during this phase of the experiment.
FIG. 17 is a screendump of an exemplary "hole test" from 48 wells
of a measurement sequence. The hole test may be used to measure
resistance by applying a small, square-wave test voltage to each
pin of the electronics head, and measuring the resultant current
from the respective trans-impedance amplifier. The individual plots
show the measured current and the resulting resistance of each
photo-machined hole in MOhms. The instrument uses these recordings
to document the quality of the multi-well substrate and to verify
that all systems are operational before proceeding with biological
measurements. The instrument also uses these recordings to verify
that each well of the multi-well substrate has effectively
"primed," thereby yielding a measurable resistance. An air bubble
present in a microhole at this point would yield an "open"
circuit," resulting in a very large resistance measurement (e.g.,
several GOhms), and as such can be used to "flag" a problem in
system performance. In addition, a statistical measure from the
"hole test" measurements may be used to correct the subsequent
voltage waveforms for voltage offsets present due to artifacts
associated with liquid junction potentials (formed at the interface
of the internal and external saline solutions) or day-to-day
variations in the absolute silver/chloride electrode
potentials.
Once the "hole test" procedure is finished, the fluidics head then
proceeds to aspirate a sufficient volume from a (previously
prepared) cell slurry reservoir to dispense approximately 3-4 .mu.l
of slurry into the top-side of every well of the multi-well
carrier. This operation is performed while a slight differential
vacuum is placed across the substrate, ensuring fluid flow (top to
bottom) through each individual photo-machined hole in the
substrate. This top to bottom flow ensures that during the sealing
process only clean "external" buffer comes into contact with the
microhole. Once the pipetting is finished, the system waits
approximately 3-5 minutes, allowing differential pressure to pull a
cell to each individual hole of the substrate and also allowing for
high-resistance seal formation to take place. Once this time period
is over, the electronics head performs a "seal test" by again
applying a square-wave voltage (typically 10 mV amplitude), and
measuring the resultant current from each respective
trans-impedance amplifier.
FIG. 18 is a screendump from 48 wells of a measurement sequence.
The average seal resistances have improved from approximately 3
MOhms during the "hole test" to approximately 150-200 MOhms during
the "seal test". This difference in resistance is due to an
individual cell lodging itself into the microhole, thereby
increasing the resistance from the top-side chamber to the bottom
side reservoir.
Following seal formation by the cell or biological membrane, the
instrument typically exchanges fluids in the plenum reservoir
beneath the substrate, replacing the high potassium internal
solution with one containing a perforation solution. This exchange
process is performed using the plenum peristaltic pump and at
static differential vacuum, so as to maintain the previously
achieved seal resistance between biological membrane and
substrate.
The presence of a commonly used antibiotic for electrical
permeabilization of the membrane has been reported to preclude the
formation of a high-resistance seal between cells and a commonly
used glass pipette. In a high-throughput system, this would require
that the initial fluids on both sides of the measurement substrate
be free from a perforation chemical solution until after a
high-resistance electrical seal forms between the biological
membrane and the measurement substrate, and that this solution be
exchanged after the electrical seal has formed in a manner that
does not disrupt the seal.
In contrast to the literature, we have found that it sometimes is
desirable and feasible to include the perforation chemical in the
initial internal solution, i.e., before a high-resistance seal has
formed. This technique has the advantage of removing an entire
fluid exchange step in the measurement protocol, as well as
initiating the chemical process of electrical access through the
cell membrane earlier in the process, greatly reducing the amount
of time required before the system can make physiological ion
channel measurements.
Typical plenum fluid exchange times are on the order of 60 seconds,
after which the fluid in the plenum is "cycled" for several
minutes. The perforation agent forms low-resistance electrical
pathways through the accessible biological membrane at each
micro-hole interface. Depending on the agent and the biological
membrane of interest, this process can take anywhere from 5 minutes
to 30 minutes to stabilize. Typical perforation times are on the
order of 10 minutes.
Once electrical access to the cell interior is achieved, the
electronics head then is able to voltage clamp the biological
membrane in each respective well, thereby enabling
electrophysiological recordings. FIG. 19 consists of an electrical
read made after achieving voltage clamp, whereby a voltage waveform
was applied that depolarized the cells from a resting potential of
-90 mV to 0 mV for approximately 100 msec. The biological membranes
under study were CHO (Chinese Hamster Ovary) cells stably
transfected with a sodium channel. As shown, upon depolarization, a
small inward current measuring on the order of a few nA (10.sup.-9
Amp) is present with the characteristic time signature of Na
channel recordings. The time scale of FIG. 19 is only 15
milliseconds wide, reflecting the fact that the Na channels, once
activated, quickly inactivate within about 3-5 milliseconds.
One measurement sequence comprises taking a pre-compound recording
from each well, followed by the addition of an experimental
compound by the fluidics head. Because of potential failures in
making recordings in each well, the system must build in a level of
redundancy to ensure that every experimental compound is tested.
Depending on the cell type and assay, typical success rates are on
the order of 50-80%. Failures may reflect a number of factors,
including imperfect substrate priming, cell debris reaching the
hole before a valid cell, and cells not having the particular ion
channel current of interest. In the preferred embodiment, to ensure
a valid measurement, a particular experimental compound is added to
multiple wells of the multi-well substrate. This provides some
system redundancy at the expense of compound throughput, i.e., the
number of different compounds that can be analyzed per day. In the
current implementation, the fluidics heads adds each well of a 96
well compound plate (or one quadrant of a 384 well compound plate)
to each of the 384 (8.times.48) wells of the measurement substrate
4 times. After each successive and distinct compound addition, the
fluidics head returns to the wash reservoir, where washing solution
is supplied to each fluidics head needle. The fluidics head
initiates a multiple aspirate/dispense cycle coincident with wash
fluid being pumped to the needle in a process that ensures that
both the inside and outside of the fluidics head needles are
cleaned effectively before grabbing the next compound set.
After a short incubation time (during which the compound is in the
presence of the biological membrane, typically 3-5 minutes), the
electronics head revisits the substrate, initiating the same
recording sequence to measure the effect of each compound on a
post-compound recording. Measurements, in this manner allow the
direct comparison of the same cell before and after the addition of
an experimental compound. This makes the measurement "differential"
in nature, allowing for good assay performance even in the presence
of widely varying individual ion channel current levels, as each
well can serve as its own control. In addition, differential
measurements offer the advantage, in the case of inhibition-type
assays, to discriminate between cells that have particular ion
channel current expression and those that do not. Cells without
expression on the pre-compound recording thus can be excluded from
post-analysis. This type of measurement sequence is only one of
many possible given the programmable functionality of the
measurement system. In some cases, the experimental compounds may
be added first, followed by a single electrical measurement read.
Some protocols rely on two fluid additions, e.g., in the case of
ligand-gated assays in which a known agonist is added to initiate a
response, followed by an electronic read, compound read, and post
read. In other cases, a second top side fluid addition can be used
to add a known control, either for normalization of prior signals
or to ensure that each well has the has the necessary ion channel
currents of interest.
The apparatus may be used using any suitable set of steps, as
determined by the cell, channel, test compound, assay format, and
so on. The remainder of this section describes without limitation a
few exemplary experimental protocols, or portions thereof, to
supplement those presented above: A. Assay 1. Pre-read, add test
compound, post-read, and compare pre-read and post-read. This
protocol may be used for typical inhibition assays, among others,
and is good for voltage-gated assays. This approach assumes that
there is a current to measure in the cell; if not, the results may
be discarded. B. Assay 2. Add agonist, pre-read, add test compound,
post-read, and compare pre-read and post-read. This protocol may be
used for slowly activated ligand-gated-channels, among others. C.
Assay 3. Add test compound, add agonist, and read. This protocol
does not use a pre-read, but assumes that most cells have a current
of known or average value. D. Assay 4. Add caged compound,
stimulate and pre-read simultaneously, add test compound, stimulate
and post-read simultaneously, compare pre-read and post-read. This
protocol may be used with caged (or activatable) compounds to
initiate a simultaneous and rapid stimulation/read of a fast
ligand-gated channel. E. Assay 5. Add caged compound, add test
compound, stimulate and post-read simultaneously. This protocol
does not use a pre-read, but again assumes that most cells have a
current of known or average value (e.g., due to known channel
expression). F. Assay 6. Pre-read, add test compound, post-read,
add known compound (agonist or antagonist), re-read. The last read
may be used for calibration of the data. III.I Software Control and
Analysis
This section describes exemplary aspects of software control and
analysis, including (1) experiment set-up and control, (2) data
analysis and reduction, and (3) leak reduction.
III.I.1 Experiment Set-up and Control
The system control software is presented as a graphical user
interface, allowing the user to program various aspects of system
control and data analysis. Examples of system control aspects
include experiment scheduling, voltage waveform definition and
timing, and compound addition scheduling and timing. FIG. 20 shows
the GUI interface for overall experiment timing, as well as data
file naming and directory designation. FIG. 21 shows the dialog box
used to establish the number of wells to be tested (expressed as a
percentage of the total), as well as the voltage waveform and
timing to be associated with the "seal test" measurement. FIG. 22
shows the set-up and timing required for the addition of the
perforation agent. FIG. 23 shows the dialog box associated with the
definition of the voltage waveform, pre-holding voltages, and
timing, as well as data sampling rates. Finally, FIG. 24 shows a
dialog box used to define the compound addition sequence, timing,
incubation time, and offset voltage corrections.
III.I.2 Data Analysis and Reduction
In addition to system control definitions, the system software also
provides the means for analyzing data. Because the measurement
process (prime, seal, measurable physiological current) does not
have a 100% probability of success in each well, the system has
built-in a level of redundancy, whereby a single experimental
compound is added to multiple (typically 4) measurement wells. The
software analysis includes in part the ability to deconvolve the
various redundant measurement wells that are associated with a
given experimental compound, analyze and scale the various time
signatures associated with pre and post compound temporal
recordings, and highlight areas of interest on the measurement
plate according to various defined metrics. As an example, FIG. 25
depicts a compound plate view in which the software has been set up
to compile the 4 individual compound measurements showing not only
how many measurements (of the 4 possible) per compound were
successful, but also to highlight according to % inhibition
(post-read to pre-read comparison) the effects of the various
compounds on the measurements. This latter feature is useful in
tracking assay integrity as every plate can include positive and
negative controls (wells which initiate a response and well that
should not) so as to verify the robustness of the particular assay.
In this particular example, FIG. 25 shows that column 11 represents
wells that show a greater than 50% inhibition of signal, whereas
column 12 represents wells that show less than 50% inhibition. This
type of view can give the user a visual and efficient quantitative
assessment of assay quality.
In addition, the user can then "drill down" by double-clicking on
each compound well to see the representative time traces that went
into the analysis. FIG. 26 is the result from one such drill down
on a "negative control well", i.e., a well which should initiate no
post-read inhibition. Shown on the plot are the pre and
post-compound time signals for the 4 associated measurement wells
corresponding to this non-active compound.
Another interesting and necessary feature of the data analysis
software is the ability to define data "metrics" which compress the
temporal signals according to user-defined mathematical expressions
into simple numbers for post-analysis processing. Such metric
definitions are essential in analyzing data from any
high-throughput instrument, as it is difficult for an end-user to
inspect hundreds of time traces generated every 30 minutes. FIG. 27
represents a metric definition window where the user has the
ability to define various simple statistical measures, such as
minimum, maximum, mean, etc., over a user-defined temporal
region(s). In the example shown, the metric is defined as the
maximum of the individual time curves between time points 20.3 and
30, as referenced to the mean of the same time curve between time
points 95 and 105. This resultant difference is saved as an
absolute value. The user then has the ability to take this simple
process number (or metric) and export it to a file that easily can
be accessed by various spreadsheet software packages for
high-throughput plate-based analysis. This type of metric
definition and processing is particularly advantageous for the
assays described here, due to the measurement throughput of the
apparatus and the inability for a user to look at all the raw time
trace information gathered per run
Another feature that is useful in the data acquisition software is
the ability to change the data sampling rates during a voltage
waveform, as well enabling user-definable "regions of interest" for
data sampling. Due to the complex nature of ion channel gating,
many assays require substantial and lengthy voltage setup
protocols. However, in many of these cases, the useful and
interesting physiological data is confined to a small interval or
fraction of the entire protocol. Having the ability to down-select
data acquisition to regions of interest greatly reduces the data
handling and storage requirements of the system. Key examples of
such protocols are those involving "use-dependent" pharmacological
protocols, whereby the action of the drug increases the more times
the channel is stimulated. During such protocols, it is not
uncommon to want to stimulate the cell many times (10-50) over
several minutes. However, the pharmacological data of interest may
only be a small fraction of the total post-compound waveform
protocol, e.g., the first pulse and the last pulse of the
stimulation train. In these cases, the user could specify a very
short and defined region in which the data is actually digitized
and subsequently saved to reduce the demands of down-stream data
handling. This also can be accomplished in part by changing data
sampling rates (or "gear shifting"), whereby regions of greater
interest are sampled more finely (e.g., at about 1 kHz) than
regions of lesser interest.
III.I.3 Leak Reduction
One of many nonobvious results from this invention was the
discovery that very successful electrophysiological recordings can
be acquired with high-resistance seals much less than a GigaOhm
(GOhm; 10.sup.9 Ohm). It generally has been accepted that, to
achieve high quality whole-cell electrophysiological recordings
from a traditional patch clamp, the leak current, i.e., the measure
of the amount of current that can flow from the sense electrode to
the ground electrode that does not flow through the cell membrane
must, be very small. When expressed as a leak resistance (applied
voltage divided by leak current), the preferred value for high
quality recordings is thought to be near 1 GOhm and is referred to
as a "GigaSeal". The reasons for this acceptance are that (i) if
uncorrected, the current can be large compared to the true
electrophysiological current, (ii) the large leak current
contributes noise to the system, and (iii) the low resistance
pathway to ground that the leak current offers greatly diminishes
the ability of the system to voltage clamp, (i.e., control the
voltage) of the biological membrane.
We have discovered that in making whole cell recordings of this
type, high-resistance seals on the order of about 20 MOhms or
higher will suffice in making quality whole-cell
electrophysiological recordings. The leak current depends on the
applied voltage waveform and the quality of the high-resistance
seal. As an example, the leak current component would be
approximately 1 nA for a 100 mV signal applied to a 100 MOhm seal.
It has been determined, however, that leak currents of a few nA do
not degrade the noise performance of a whole-cell clamp
significantly, assuming that one can isolate the leak component
from the rest of the signal. Furthermore, while the effect on the
voltage clamp does create a voltage clamp error, such error is
acceptable in most whole-cell recordings. For high-resistance
seals, on the order of 100 MOhms, the error in voltage clamp as
compared to that achieved under a GigaSeal is on the order of a few
mV. Errors of a few millivolts have little impact on the
performance of the system.
The biggest drawback to have large leak currents has to do with the
ability to establish a true "baseline" from which to isolate the
physiological current from the biological membrane from that of the
leak component. It has been discovered, that in most cases with
proper signal design, that the effects of the leak current can be
corrected. FIG. 28 demonstrates two electrophysiological traces,
with and without leak correction. Both signals were obtained using
a command voltage having a depolarization step to +50 mV at time
point 20 msec using the delayed rectifying potassium channel Kv1.5,
after the application of the time-dependent blocking compound,
tedisamil. As shown, depolarization step causes a time-decaying
physiological current over the depolarization pulse. To correct the
"leak current" component, the command voltage waveform also
contains a "pre-pulse," comprising a small step voltage from -80 to
-70 mV. (The pre-pulse may be conducted over any suitable voltage
range, typically in the physiological (-100 to +60 mV)
physiological range, in which there is little or no physiological
current.) Relying on the fact that there is no physiological
current triggered at these negative voltages, the measured current
resulting from this pre-pulse can be used to isolate and
subsequently estimate the leak current. This estimated value can
then be used to "subtract" off the effects of the leak current
during the entire voltage waveform, thereby yielding a true
estimate of the baseline physiological current. Using this
technique, we have been able to greatly increase the probability of
success in making high-throughput electrophysiological recordings,
by effectively lowering the acceptable high-resistance threshold
from GOhms to tens of MOhms.
IV. Channel/Transporter Assays
The invention provides systems, including apparatus and methods,
for monitoring the influence of effector agents and their
modulators on membrane electrical activity. The effector agents may
include activating/stimulatory agents and/or
deactivating/inhibitory agents, among others, and modulators
thereof. The system may be used to study any suitable
electrophysiological process or event, particularly those involving
ligand-gated ion channels and/or transporters. The system may be
used with single samples, for example, using pipette-based or
planar-substrate-based measurement devices. However, preferably,
the invention may be used with multiple samples, sequentially
and/or simultaneously, thereby enabling the study of fast
ligand-gated electrophysiological events in a high-throughput
manner.
IV.A Ion Channels/Transporters
The apparatus and methods provided by the invention may be used to
study membrane components that are associated with or capable of
bringing about measurable voltage changes and/or current flows
across biological membranes. Suitable membrane components may
include ion channels and ion transporters, among others,
particularly ligand-gated channels and transporters.
IV.A.1 Ion Channels
Ion channels are membrane proteins that allow ions to flow across
biological membranes, including the plasma membrane and organelle
membranes. Ion channels are believed to create water-filled pores
through which ions and some small hydrophilic molecules can pass by
diffusion (i.e., the associated ion flow is passive, meaning that
it occurs down a electrochemical gradient without requiring the
input of energy.) Ligand-gated channels open or close in response
to the binding, reaction, and/or other association of signaling
molecules, termed "ligands." These channels may be gated by the
binding of extracellular or intracellular ligands. In either case,
the ligand is different than the substance that is transported when
the channel opens. IV.A.1.a Externally Gated Ion Channels
External ligands gate a variety of ion channels, including (1) ATP
gated-channels, (2) glutamate-activated cationic channels, and (3)
cys-loop superfamily channels. The ATP-gated channel superfamily
includes the ATP2x and ATP2z receptors, among others. The
glutamate-activated cationic receptor superfamily includes the
NMDA, AMPA, and Kainate receptors, among others. Finally, the
cys-loop receptor superfamily includes the nicotinic acetylcholine
receptor, GABA.sub.A and GABA.sub.C receptors, glycine receptors,
5-HT.sub.3 receptors, and anionic glutamate receptors, among
others. These particular channels are controlled by the ligands
that appear in the names of the channels.
External ligands most often are neurotransmitters, that is,
chemical substances that transmit nerve impulses across a synapse,
typically to another nerve cell or a muscle cell. Exemplary
neurotransmitters include acetylcholine (Ach), amino acids (e.g.,
glutamic acid (Glu), glycine (Gly), and gamma aminobutyric acid
(GABA)), catecholamines (e.g., noradrenaline and dopamine),
miscellaneous monoamines (e.g., serotonin and histamine), and
peptides (e.g., vasopressin (ADH), oxytocin, Gonadotropin-releasing
hormone (GnRH), angiotensin II, cholecystokinin (CCK), substance P,
and enkephalins such as Met-enkephalin and Leu-enkephalin), among
others. These transmitters interact in the body with channels in
the postsynaptic membrane to depolarize or hyperpolarize the
postsynaptic membrane, depending on the transmitter and on whether
the synapse is excitatory or inhibitory, respectively. IV.A.1.b
Internally Gated Ion Channels
Internal ligands also gate a variety of ion channels. These
channels may include G-protein coupled receptors (GPCRs), chloride
channels, and calcium-gated potassium channels, among others. These
channels generally are controlled by second messengers, which are
small signaling molecules such as cyclic AMP (cAMP), cyclic GMP
(cGMP), and Ca.sup.2+, among others. However, some of these
channels are controlled by covalent modification, e.g.,
phosphorylation/dephosphorylation by kinases and phosphatases,
respectively.
IV.A.2 Ion Transporters
Ion transporters are membrane proteins that use energy such as that
derived from ATP to force ions or small molecules though the
membrane up their electrochemical gradients. The transporters may
be (1) direct active transporters, binding ATP directly and using
the energy of its hydrolysis to drive active transport, or (2)
indirect active transporters, using ATP indirectly by using the
downhill flow of a different type of ion to drive active transport,
where the gradient of the different type of ion is created by a
direct active transporter, allowing another transporter to create a
gradient of a different type of ion, and then using. Indirect
transporters may be further subdivided into symporters and
antiporters depending on whether the driving ion and the pumped ion
(or other molecule) pass through the membrane in the same or
opposite directions, respectively. Exemplary direct active
transporters include the Na.sup.+/K.sup.+ ATPase and the H+ ATPase.
Exemplary indirect active transporters include (1) symporters such
as the Na+/glucose transporter, the various amino acid/Na+
transporters, and the Na+/iodide transporter, and (2) antiporters
such as the Na.sup.+/K.sup.+ ATPase.
IV.B Activatable Compounds
Activatable compounds generally comprise any compounds, such as
channel and/or transporter ligands, and modulators thereof, whose
spatial and/or temporal release may be rapidly modulated by a
suitable trigger, such as a change in light and/or voltage, among
others.
Photoactivatable compounds, which are triggered by light, are
preferred for many applications. Photoactivatable compounds are
chemicals that are chemically altered such that the active nature
of the compound is suppressed ("caged") until photoactivated,
usually by a short pulse of ultra-violet (UV) light of wavelength
in the range of 240 and 400 nm. The photolysis of such compounds is
very fast and thereby can rapidly (in some cases in microseconds)
release the active species of the compound. Suitable methods for
producing these compounds and exemplary embodiments thereof are
described in the following publication, which is incorporated
herein by reference in its entirety for all purposes: Richard P.
Haugland, HANDBOOK OF FLUORESCENT PROBES AND RESEARCH CHEMICALS
(6.sup.th ed. 1996).
Photoactivatable compounds may be produced by derivatizing a ligand
or modulator or other compound of interest with one or more
photolabile protecting or caging groups. These caging groups, which
collectively form a caging moiety, are selected and/or designed to
interfere maximally with the binding, activity, and/or other
function(s) of the derivatized compound. These groups may be
detached rapidly (e.g., in microseconds to milliseconds) by
appropriate illumination (e.g., flash photolysis at .ltoreq.360
nm). The groups may be incorporated into biologically active
molecules using any suitable mechanism, for example, by linkage to
a hetero-atom (e.g., O, S, or N) as an ether, thioether, ester
(including phosphate or thiophosphate esters), amine, or similar
functional group. Exemplary caging groups may include (1)
.alpha.-carboxy-2-nitrobenzyl (CNB) groups, (2)
1-(2-nitrophenyl)ethyl (NPE) groups, (3)
4,5-dimethoxy-2-nitrobenzyl (DMNB) groups, (4)
1-(4,5-dimethoxy-2-nitrophenyl)ethyl (DMNPE) groups, and (5)
5-carboxymethoxy-2-nitrobenzyl (CMNB) groups, among others.
Significantly, when intracellular application is required, the
caged compound often can be made cell permeable, such that it can
be loaded into the cytoplasm of the cell for rapid intracellular
activation at a later time.
Suitable photoactivatable compounds may include appropriately caged
ligands, caged modulators, and the like, depending on the assay.
Exemplary caged ligands include caged neurotransmitters and caged
second messengers. Commercially available caged neurotransmitters
include caged carbamylcholine, caged .gamma.-aminobutyric acid
(GABA), caged N-methyl-D-aspartic acid, and caged L-glutamic acid,
all of which are biologically inactive before photolysis (Molecular
Probes, Eugene, Oreg., USA). Commercially available caged second
messengers include caged cAMP, caged inositol 1,4,5-triphosphate,
caged cADP-ribose, and caged Ca.sup.2+, at least several of which
are membrane permeant (Molecular Probes). Exemplary caged
modulators include caged ligand chelators, which can bind up ligand
already present so that it no longer can bind to channels.
Commercially available caged ligand chelators include caged
Ca.sup.2+ chelators (Molecular Probes).
IV.C Assays
The invention provides among others electrophysiological assays
involving the use of activatable compounds, particularly for the
study of ligand-gated membrane components such as ligand-gated
channels and transporters. Activatable compounds may be especially
useful in high-throughput applications, because they can be used to
"introduce" compounds into solution, near an appropriate receptor,
without requiring that the compound be pipetted into the solution
at the time of the electrical measurement. This capability may be
especially useful in systems such as the specific embodiment
described above, in which rapid introduction or perfusion, on the
time scale of typical channel or transporter kinetics, is
difficult.
The assays may have any suitable design. Typically, caged versions
of a ligand or modulator will be introduced into a system, and then
activated at a suitable time using a suitable trigger, such as
application of light. The electrical activity of the sample may be
measured before, during, and/or after activation, so that the
kinetic effects of the uncaged compound on the phenomenon of
interest can be studied. Thus, in some assays, the caged compound
may be a caged ligand, with the assay monitoring the effects of the
ligand on a channel or transporter, typically in the presence of a
candidate modulator. In other assays, the caged compound may be a
caged ligand chelator or caged ligand degrader, with the assay
monitoring the effects of removing the ligand from a system
potentially habituated to the ligand, for example, by binding it up
or destroying it. In yet other assays, the caged compound may be a
caged modulator, with the assay monitoring the effects of the
modulator on a system already exposed to the ligand.
V. EXAMPLES
The following numbered paragraphs describe additional and/or
alternative aspects of the invention:
1. Electrophysiological measurement apparatus, comprising (A) a
measurement platform including a moveable electronics head equipped
with one or more sensing electrodes in communication with a signal
processor and output device, and a moveable fluidics head equipped
with one or more fluid-dispensing needles; (B) the platform
including a plurality of stations, at least one of the stations
being an integrated measurement plenum accessible by both the
electronics head and the fluidics head, the measurement plenum
including (i) a multi-well plate having a plurality of
fluid-carrying chambers, each chamber containing biological
material under investigation, and (ii) a thin substrate having an
array of apertures in alignment with the chambers of the multi-well
plate; and (C) a fluidics system operative to control and regulate
the differential pressure across the substrate to achieve a
high-resistance electrical seal between the substrate and the
biological material.
2. The high-throughput electrophysiological measurement apparatus
of paragraph 1, wherein the fluidics system is further operative to
remove trapped gas from both sides of the substrate to form a
continuous fluid pathway for conducting electrical current.
3. The high-throughput electrophysiological measurement apparatus
of paragraph 1, wherein the fluidics system is further operative to
apply a vacuum so as to pneumatically isolate a region on one side
of the substrate.
4. The high-throughput electrophysiological measurement apparatus
of paragraph 1, further including reagent inputs for one or more of
the following: an extracellular saline solution, intracellular
saline solution, wash solution, and chemically altered
intracellular saline solution used to achieve low resistance
electrical access to the inside of a cell.
5. The high-throughput electrophysiological measurement apparatus
of paragraph 4, wherein intracellular solutions are exchanged
without introducing pressure changes that would disrupt the
high-resistance electrical seal.
6. The high-throughput electrophysiological measurement apparatus
of paragraph 1, wherein the signal processor includes a multiplexer
operative to route electrical signals derived from one or more of
the electrodes on a selective basis.
7. The high-throughput electrophysiological measurement apparatus
of paragraph 1, wherein the fluidics head includes a multi-channel
manifold segment and a spatially offset single channel segment
facilitating multi-channel and single channel operation.
8. The high-throughput electrophysiological measurement apparatus
of paragraph 1, wherein the signal processor includes a low-noise,
high-gain trans-impedance operational amplifier circuit and one or
more isolated recording amplifier circuits.
9. The high-throughput electrophysiological measurement apparatus
of paragraph 1, further including one or more positions for washing
for the electronics head and the fluidics head.
10. The high-throughput electrophysiological measurement apparatus
of paragraph 9, wherein each position for washing is automatically
filled and drained via a peristaltic pump and vacuum assisted waste
line.
11. The high-throughput electrophysiological measurement apparatus
of paragraph 9, wherein each position for washing is capable of
washing an entire head or portions thereof to conserve conserving
wash solution.
12. The high-throughput electrophysiological measurement apparatus
of paragraph 1, wherein the stations include one or more compound
microplates, each having a standard well format accessible by the
fluidics head.
13. The high-throughput electrophysiological measurement apparatus
of paragraph 12, wherein the stations include one or more saline
solution reservoirs accessible by the fluidics head.
14. The high-throughput electrophysiological measurement apparatus
of paragraph 1, wherein the stations include a removable boat
position for cell slurry addition accessible by the fluidics
head.
15. The high-throughput electrophysiological measurement apparatus
of paragraph 1, wherein the electronics head uses silver sensing
electrodes, and the apparatus includes a station accessible by the
electronics head used for depositing chloride on the
electrodes.
16. A fluidics apparatus, comprising (A) a fluidics head
operatively positioned to provide fluid to an examination site; and
(B) a first dispenser set and a second dispenser set, both
dispenser sets being connected to the fluidics head, wherein the
dispenser sets are spaced from each other such that fluid can be
alternately delivered exclusively from the first dispenser set, and
exclusively from the second dispenser set, to a multi-compartment
sample holder located at the examination site.
17. The apparatus of paragraph 16, wherein multiple samples at the
examination site may be arranged across a plane, a Z axis being
defined perpendicular to the plane, the first and second dispense
sets being positioned at different heights relative to the Z
axis.
18. The apparatus of paragraph 17, wherein the second dispense set
has a single dispenser element positioned lower than the first
dispense set relative to the Z axis, so that when the single
dispenser element is dispensing fluid to a sample at the
examination site, the second dispense set avoids contact with any
other samples at the examination site.
19. The apparatus of paragraph 16, wherein the first dispense set
includes multiple dispenser elements, and the second dispense set
includes a single dispenser element.
20. The apparatus of paragraph 16, wherein each of the first and
second dispense sets includes multiple dispenser elements.
21. An apparatus for measuring an electric property of a membranous
sample, comprising (A) a platform; (B) an examination station on
the platform; (C) a container at the examination station including
first and second compartments separated by a substrate having one
or more apertures arranged and dimensioned for conducting a patch
clamp experiment on a membranous sample in the first compartment;
(D) one or more sample processing stations on the platform; (E) a
fluidic transfer device mounted on the platform configured to move
material from the reservoir station to the container at the
examination station; and (F) an electrode device configured to move
in and out of contact with the membranous sample in the first
compartment of the container at the examination station.
22. The apparatus of paragraph 21, wherein the sample processing
stations and the examination station are arranged along a linear
processing path, so that the fluidic transfer device and the
electrode device can move between stations along overlapping
segments of the processing path.
23. The apparatus of paragraph 22, processing path has a detection
segment and a preparation segment, the detection segment and the
preparation segment overlapping in a region including the
examination station.
24. The apparatus of paragraph 21, wherein the sample processing
stations include one or more input stations for holding sample or
reagent material to be transferred to the examination station.
25. The apparatus of paragraph 21, wherein the sample processing
stations include one or more wash stations.
26. The apparatus of paragraph 21, wherein the sample processing
stations include one or more renewal stations.
27. The apparatus of paragraph 21, further comprising a computer,
program, and interface configured to allow a user to determine a
processing routine including coordinated movement of the fluidic
transfer device and the electrode device relative to the
examination station.
28. The apparatus of paragraph 21, wherein the processing path has
a first end portion configured for carrying out sample preparation,
and a second end portion configured for carrying out sample
analysis.
29. The apparatus of paragraph 28, wherein the first end portion
and the second portion each include the examination station.
30. The apparatus of paragraph 21, wherein each of the fluidic
transfer device and the electrode device has a portion that is
moveable along a Z axis perpendicular to the platform.
31. An apparatus for conducting a biological experiment, comprising
(A) a platform; (B) an examination station located on the platform
along a linear processing path; (C) one or more sample preparation
stations located along the processing path; (D) a guide rail
structure mounted on the platform substantially parallel to the
processing path; (E) a fluidics head moveable along the guide rail
for transferring material between stations along the processing
path; and (F) a detector head moveable along the guide rail,
configured to sense a property of a biological sample located at
the examination station.
32. The apparatus of paragraph 31, further comprising a container
at the examination station including first and second compartments
separated by a substrate having one or more apertures arranged and
dimensioned for conducting a patch clamp experiment on a membranous
sample in the first compartment.
33. The apparatus of paragraph 21, wherein a first electrode is
connected to the second compartment, the detector head having a
second electrode for contacting the membranous sample in the first
compartment.
34. The apparatus of paragraph 31, further comprising a drive
mechanism that drives each of the fluidics head and the detector
head along the guide rail.
35. The apparatus of paragraph 31, further comprising (G) a first
drive mechanism that drives the fluidics head along the guide rail,
and (H) a second drive mechanism that drives the detector head
along the guide rail.
36. The apparatus of paragraph 31, wherein the fluidic transfer
device and the electrode device can move between stations along
overlapping segments of the processing path.
37. The apparatus of paragraph 31, wherein the processing path has
a detection segment and a preparation segment, the detection
segment and the preparation segment overlapping in a region
including the examination station.
38. The apparatus of paragraph 31, wherein the one or more sample
processing stations include one or more input stations for holding
sample or reagent material to be transferred to the examination
station.
39. The apparatus of paragraph 31, further comprising a computer,
program, and interface configured to allow a user to determine a
processing routine including coordinated movement of the fluidic
transfer device and the electrode device relative to the
examination station.
40. A method for measuring an electrical property of a membranous
sample, comprising (A) selecting an electrophysiological
measurement apparatus comprising (i) a substrate having an
aperture, (ii) first and second fluid compartments, separated by
the substrate, in fluid communication via the aperture, and (iii)
first and second electrodes, the first electrode in electrical
contact with the first fluid compartment, and the second electrode
in electrical contact with the second fluid compartment; (B) adding
a perforation agent to the second compartment, the perforation
agent being capable of forming an electrically conductive hole
through the membranous sample; (C) adding the membranous sample to
the first compartment, after the step of adding a perforation agent
to the second compartment; (D) sealing the membranous sample across
the aperture to form an electrically tight seal; and (E) measuring,
using the electrodes, at least one of a current and a voltage
across the aperture and at least a portion of the membranous
sample.
41. The method of paragraph 40, the membranous sample including a
trapped volume, wherein the perforation agent forms an electrically
conductive hole through the membranous sample, such that the
trapped volume is in fluid communication with the second fluid
compartment but not with the first fluid compartment.
42. The method of paragraph 40, wherein the perforation agent is
selected from the group consisting of amphotericin B and
nystatin.
43. An electrophysiological measurement apparatus, comprising (A) a
sample holder having a plurality of electrophysiological
measurement sites; (B) a plurality of electrodes, each electrode in
electrical contact with a different measurement site; and (C) a
controller configured to set the electrodes for one of at least two
functions, and to switch the electrodes between the at least two
functions; wherein the controller has set a first set of electrodes
for a first function, and a second set of electrodes for a second
function, the first and second functions being different.
44. The method of paragraph 43, wherein the controller changes the
function performed by the first set of electrodes from the first
function to the second function.
45. The method of paragraph 43, wherein the controller interchanges
the functions performed by the two sets of electrodes.
46. The method of paragraph 43, wherein the first function is to
hold a voltage across a sample at the measurement site at a
preselected value, and wherein the second function is to measure an
electrical property of a sample at the measurement site.
47. The method of paragraph 46, wherein the electrical property is
at least one of a current or a voltage across at least a portion of
the sample.
48. The method of paragraph 43, wherein the controller includes a
multiplexer configured to route electrical signals derived from the
electrodes on a selective basis.
49. The method of paragraph 43, wherein the measurement sites
comprise a substrate having an aperture, and first and second fluid
compartments, separated by the substrate, in fluid contact via the
aperture.
50. The method of paragraph 43, wherein the controller includes a
low-noise, high-gain, trans-impedance operational amplifier circuit
and at least one isolated recording amplifier circuit.
51. The method of paragraph 43, wherein the first function is
performed by an analog-to-digital (A/D) converter.
52. The method of paragraph 43, wherein the electrodes are disposed
in an electronics head, and wherein the electronics head is
moveable so that the electrodes can be moved to bring them into
contact with a different set of measurement sites within the sample
holder.
53. The method of paragraph 43, the apparatus including 48
electrodes, wherein 36 of the electrodes are set to perform the
first function, and wherein 12 of the electrodes are set to perform
the second function.
54. A method of performing an electrophysiological experiment,
comprising (A) positioning a plurality of samples at a
corresponding plurality of measurement sites, in an
electrophysiological measurement apparatus; (B) positioning a
plurality of electrodes at least a subset of the measurement sites,
each electrode in electrical contact with a different measurement
site; (C) setting a first set of electrodes to perform a first
function; (D) setting a second set of electrodes to perform a
second function, wherein the second function is different than the
first function; and (E) changing the function performed by the
first set of electrodes from the first function to the second
function.
55. The method of paragraph 54, further comprising changing the
function performed by the second set of electrodes from the second
function to the first function.
56. The method of paragraph 55, wherein the steps of changing the
function performed by the first set of electrodes and the function
performed by the second set of electrodes are performed
simultaneously.
57. The method of paragraph 54, further comprising setting the
first and second set of electrodes to perform the same
function.
58. The method of paragraph 54, wherein the first function is to
hold a voltage across a sample at the measurement site at a
preselected value, and wherein the second function is to measure an
electrical property of a sample at the measurement site.
59. The method of paragraph 58, wherein the electrical property is
at least one of a current or a voltage across at least a portion of
the sample.
60. The method of paragraph 54, wherein the first function is
performed by an analog-to-digital (A/D) converter.
61. The method of paragraph 54, further comprising moving the
electrodes to bring them into contact with a different set of
measurement sites within the sample holder.
62. The method of paragraph 61, further comprising repeating, for
the different set of measurement sites, the steps of setting the
first set of electrodes to perform a first function, setting the
second set of electrodes to perform the second function, and
changing the function performed by the first set of electrodes from
the first function to the second function.
63. The method of paragraph 54, there being 48 electrodes, wherein
36 of the electrodes are set to perform the first function, and
wherein 12 of the electrodes are set to perform the second
function.
64. A method for measuring an electrical property of a membranous
sample, the membranous sample enclosing a trapped volume, the
method comprising (A) selecting an electrophysiological measurement
apparatus comprising (i) a substrate having an aperture, (ii) first
and second fluid compartments, separated by the substrate, in fluid
communication via the aperture, and (iii) first and second
electrodes, the first electrode in electrical contact with the
first fluid compartment, and the second electrode in electrical
contact with the second fluid compartment; (B) adding the
membranous sample to the first fluid compartment, the membranous
sample forming an electrically tight seal across the aperture; (C)
permeabilizing at least a portion of the membranous sample, after
it is sealed across the aperture, such that the trapped volume is
in fluid communication with the second fluid compartment but not
with the first fluid compartment; (D) exchanging at least a portion
of the fluid in the second fluid compartment, while maintaining the
electrically tight seal; and (E) measuring, using the electrodes,
at least one of a current and a voltage across the aperture and at
least a portion of the membranous sample.
65. The method of paragraph 64, wherein the membranous sample is
selected from the group consisting of cells, subcellular
organelles, and vesicles.
66. The method of paragraph 64, wherein the electrophysiological
measurement apparatus further comprises an automated fluid exchange
system configured to exchange fluid in the second compartment.
67. The method of paragraph 66, wherein the fluid exchange system
maintains the second fluid compartment at a slightly lower pressure
than the first fluid compartment.
68. The method of paragraph 64, wherein the step of measuring at
least one of a current and a voltage is performed before and after
the step of exchanging at least a portion of the fluid in the
second fluid compartment.
69. The method of paragraph 64, wherein the step of permeabilizing
the portion of the membranous sample sealed across the aperture
includes contacting the portion with a pore former.
70. The method of paragraph 64, wherein the step of permeabilizing
the portion of the membranous sample sealed across the aperture
includes disrupting the portion using an electrical pulse.
71. The method of paragraph 64, wherein the step of permeabilizing
the portion of the membranous sample sealed across the aperture
includes disrupting the portion using a pressure pulse.
72. The method of paragraph 64, wherein the step of exchanging at
least a portion of the fluid in the second fluid compartment
results in the exchange of at least a portion of the fluid in the
trapped volume.
73. The method of paragraph 64, wherein the portion of the
substrate adjacent the aperture is at least substantially
planar.
74. The method of paragraph 64, the electrophysiological
measurement apparatus further comprising (1) a second aperture, (2)
third and fourth fluid compartments, separated by the substrate, in
fluid communication via the second aperture, and (3) third and
fourth electrodes, the third electrode in electrical contact with
the third fluid compartment, the fourth electrode in electrical
contact with the fourth fluid compartment, further comprising (A)
adding a second membranous sample to the third fluid compartment,
such that the second membranous sample forms an electrically tight
seal across the second aperture; (B) permeabilizing at least a
portion of the membranous sample sealed across the aperture, such
that the trapped volume is in fluid communication with the second
fluid compartment; (C) exchanging at least a portion of the fluid
in the second fluid compartment, while maintaining the electrically
tight seal; and (D) measuring, using the electrodes, at least one
of a current and a voltage across the aperture and at least a
portion of the membranous sample.
75. The method of paragraph 74, wherein the second and fourth fluid
compartments are joined to form a single fluid compartment, and
wherein the second and fourth electrodes are the same.
76. Electrophysiological measurement apparatus, comprising (A) a
multi-well plate having a plurality of fluid chambers, each
configured to support a biological material to be measured; (B) a
thin substrate having an array of apertures in alignment with the
chambers of the multi-well plate, wherein the substrate is joined
to the multi-well plate such that the chambers are open at the top
and sealed at the bottom, except for the apertures, and wherein the
diameter of the apertures is less than the diameter of the
biological material, thereby enabling a high-resistance seal to be
formed between the biological material in each chamber and a
corresponding aperture; (C) a fluid plenum to receive the
multi-well plate such that one side of the substrate is immersed;
(D) a first electrode disposed in the fluid plenum; (E) at least
one second electrode moveable into the top openings of the fluid
chambers of the multi-well plate; and (F) electrophysiological
measurement circuitry in electrical communication with the
electrodes.
77. The electrophysiological measurement apparatus of paragraph 76,
wherein there is a single aperture associated with each chamber of
the multi-well plate.
78. The electrophysiological measurement apparatus of paragraph 76,
wherein each compartment contains a biological material to be
measured.
79. The electrophysiological measurement apparatus of paragraph 76,
wherein the substrate is a plastic substrate having a glass coating
at least in the region where the high-resistance seal is formed
between the material and the substrate.
80. The electrophysiological measurement apparatus of paragraph 79,
wherein the substrate is selected from the group consisting of
polyethylene terephthalate (PET) and polyimide.
81. The electrophysiological measurement apparatus of paragraph 76,
wherein the diameter of the apertures is in the range of about 1 to
10 micrometers.
82. The electrophysiological measurement apparatus of paragraph 76,
wherein the apertures are tapered.
83. The electrophysiological measurement apparatus of paragraph 76,
wherein the multi-well plate is sealed to the fluid plenum,
enabling a differential pressure to be applied relative to the
fluid in each chamber, thereby causing the material in each chamber
to migrate to a respective aperture.
84. The electrophysiological measurement apparatus of paragraph 76,
wherein the multi-well plate is sealed to the fluid plenum,
enabling a differential pressure to be maintained relative to the
fluid in each chamber until between the material in each chamber
forms the high-resistance seal to the corresponding aperture.
85. The electrophysiological measurement apparatus of paragraph 76,
wherein the fluid plenum includes a chemical reagent causing the
material in each chamber to electrically permeabilize in the
vicinity of the aperture.
86. The electrophysiological measurement apparatus of paragraph 76,
wherein a high voltage is temporarily applied across the electrodes
to permeabilize the material in each chamber, at least in the
vicinity of the apertures.
87. The electrophysiological measurement apparatus of paragraph 76,
further comprising a mechanism for moving the electrode into the
chambers of the multi-well plate so as to automate the measurement
of the material contained therein.
88. The electrophysiological measurement apparatus of paragraph 76,
further comprising (G) a plurality of electrodes in alignment with
a plurality of the chambers of the multi-well plate; and (H) a
mechanism for moving the electrodes into the chambers of the
multi-well plate to perform simultaneous measurements on the
material contained therein.
89. The electrophysiological measurement apparatus of paragraph 76,
further comprising a system for transferring fluids from one or
more sources to the chambers of the multi-well plate.
The disclosure set forth above may encompass multiple distinct
inventions with independent utility. While each of these inventions
has been disclosed in its preferred form, the specific embodiments
thereof as disclosed and illustrated herein are not to be
considered in a limiting sense as numerous variations are possible.
The subject matter of the inventions includes all novel and
nonobvious combinations and subcombinations of the various
elements, features, functions and/or properties disclosed herein.
Similarly, where the claims recite "a" or "a first" element or the
equivalent thereof, such claims should be understood to include
incorporation of one or more such elements, neither requiring nor
excluding two or more such elements. It is believed that the
following claims particularly point out certain combinations and
subcombinations that are directed to one of the disclosed
inventions and are novel and nonobvious. Inventions embodied in
other combinations and subcombinations of features, functions,
elements and/or properties may be claimed through amendment of the
present claims or presentation of new claims in this or a related
application. Such amended or new claims, whether they are directed
to a different invention or directed to the same invention, whether
different, broader, narrower or equal in scope to the original
claims, are also regarded as included within the subject matter of
the inventions of the present disclosure.
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